Treating inflammation with IL-17/IL-23 bispecific antibodies

ABSTRACT

The present invention relates to blocking, inhibiting, reducing, antagonizing or neutralizing the activity of IL-17A, IL-17F, and IL-23. Antagonists include antibodies and antibody fragments that bind IL-23 and that bind IL-17A or IL-17F, such as antibodies that are cross-reactive for IL-17A and Il-17F. Antagonists that include an antibody or antibody fragment that binds IL-23 and an antibody or antibody fragment that binds IL-17A or IL-17F on one molecule are also disclosed. Antibodies and antibody fragments that bind IL-23 and IL-17F but that do not bind IL-17A are also disclosed. IL-17 and IL-23 are cytokines that are involved in inflammatory processes and human disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/260,059, filed Sep. 8, 2016, now U.S. Pat. No. 9,994,634, which is adivisional of U.S. patent application Ser. No. 14/626,959, filed Feb.20, 2015, now U.S. Pat. No. 9,464,134, which is a divisional of U.S.patent application Ser. No. 13/929,226, filed Jun. 27, 2013, now U.S.Pat. No. 8,992,922, which is a continuation of U.S. application Ser. No.13/534,019, filed Jun. 27, 2012, now U.S. Pat. No. 8,496,936, which is adivisional of U.S. application Ser. No. 13/032,501, filed Feb. 22, 2011,now U.S. Pat. No. 8,333,968, which is a divisional of U.S. applicationSer. No. 12/111,117, filed Apr. 28, 2008, now U.S. Pat. No. 7,910,703,which claims the benefit of U.S. Provisional Application Ser. No.60/914,681, filed Apr. 27, 2007, and U.S. Provisional Application Ser.No. 60/914,663, filed Apr. 27, 2007, all of which are hereinincorporated by reference. U.S. application Ser. No. 12/111,117, nowU.S. Pat. No. 7,910,703, is a continuation-in-part of U.S. applicationSer. No. 11/762,738, filed Jun. 13, 2007, now U.S. Pat. No. 7,790,862,which claims the benefit of U.S. Provisional Application Ser. No.60/804,602, filed Jun. 13, 2006, U.S. Provisional Application Ser. No.60/824,665, filed Sep. 6, 2006, U.S. Provisional Application Ser. No.60/828,277, filed Oct. 5, 2006, and U.S. Provisional Application Ser.No. 60/891,410, filed Feb. 23, 2007, all of which are hereinincorporated by reference. U.S. application Ser. No. 12/111,117, nowU.S. Pat. No. 7,910,703, is also a continuation-in-part of U.S.application Ser. No. 11/741,189, filed Apr. 27, 2007, now U.S. Pat. No.7,790,163, which is a continuation-in-part of U.S. application Ser. No.11/684,907, filed Mar. 12, 2007, now abandoned, which claims the benefitof U.S. Provisional Application Ser. No. 60/781,121, filed Mar. 10,2006, U.S. Provisional Application Ser. No. 60/828,271, filed Oct. 5,2006, and U.S. Provisional Application Ser. No. 60/862,501, filed Oct.23, 2006, all of which are herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates generally to the identification andisolation of antagonists to IL-17A and IL-17F and IL-23 (via p19) andmethods of using the same.

BACKGROUND OF THE INVENTION

Cytokines are soluble, small proteins that mediate a variety ofbiological effects, including the induction of immune cellproliferation, development, differentiation, and/or migration, as wellas the regulation of the growth and differentiation of many cell types(see, for example, Arai et al., Annu. Rev. Biochem. 59:783 (1990);Mosmann, Curr. Opin. Immunol. 3:311 (1991); Paul and Seder, Cell 76:241(1994)). Cytokine-induced immune functions can also include aninflammatory response, characterized by a systemic or local accumulationof immune cells. Although they do have host-protective effects, theseimmune responses can produce pathological consequences when the responseinvolves excessive and/or chronic inflammation, as in autoimmunedisorders (such as multiple sclerosis) and cancer/neoplastic diseases(Oppenheim and Feldmann (eds.) Cytokine Reference, Academic Press, SanDiego, Calif. (2001); von Andrian and Mackay New Engl. J. Med. 343: 1020(2000); Davidson and Diamond, New Engl. J. Med. 345:340 (2001); Lu etal, Mol. Cancer Res. 4:221(2006); Dalgleish and O'Byrne, Cancer TreatRes. 130:1 (2006)).

IL-17A, IL-17F and IL-23 are cytokines involved in inflammation. Humaninterleukin-17A (also known as “IL-17A”) is a cytokine which stimulatesthe expression of interleukin-6 (IL-6), intracellular adhesion molecule1 (ICAM-1), interleukin-8 (IL-8), granulocyte macrophagecolony-stimulating factor (GM-CSF), and prostaglandin E2 expression, andplays a role in the preferential maturation of CD34+ hematopoieticprecursors into neutrophils (Yao et al., J. Immunol. 155:5483 (1995);Fossiez et al., J. Exp. Med. 183:2593 (1996)). Human interleukin-23(also known as “IL-23”) is a cytokine which has been reported to promotethe proliferation of T cells, in particular memory T cells.

IL-17A and IL-17F share 55% identity (Kolls and Linden, 2004). Inaddition to their sequence similarity, both of these cytokines seem areproduced by similar cell types, most notably activated, memory CD4+ Tcells. See e.g. Agarwal et al., “Interleukin-23 promotes a distinct CD4T cell activation state characterized by the production ofinterleukin-17” J. Biol. Chem. 278:1910-191 (2003); see also Langrish etal. “IL-23 drives a pathogenic T cell population that induces autoimmuneinflammation” J. Exp. Med. 201: 233-240 (2005); and Starnes et al.“Cutting edge: IL-17F, a novel cytokine selectively expressed inactivated T cells and monocytes, regulates angiogenesis and endothelialcell cytokine production” J. Immunol. 167:4137-4140 (2001).

While IL-17F shares sequence homology with IL-17A, there are keydifference between these molecules. For example, IL-17F mRNA is detectedin many different tissues (such as, liver, lung, ovary, fetal liver,mast cells and basophils) while IL-17A expression is mostly restrictedto T cells. See Fossiez, F., et al., “T cell IL-17 induces stromal cellsto produce pro-inflammatory and hematopoietic cytokines”, J. Exp. Med.183(6):2593-2603, (1996); Toy, D. et al., “Cutting edge: IL-17 signalsthrough a heteodimeric receptor complex”, J. Immunol.177(1):36-39(2006). Additionally, IL-17F binds IL-17RA with a much loweraffinity than IL-17A.

Moreover, both have been similarly implicated as contributing agents toprogression and pathology of a variety of inflammatory and auto-immunediseases in humans and in mouse models of human diseases. Specifically,IL-17A and IL-17F have been implicated as major effector cytokines thattrigger inflammatory responses and thereby contribute to a number ofautoinflammatory diseases including multiple sclerosis, rheumatoidarthritis, and inflammatory bowel diseases.

Recently it has been shown that using a combination of an antagonist toIL-17A and an antagonist to IL-23, relapse was prevented in a mousemodel of multiple sclerosis. See co-owned U.S. patent application Ser.No. 11/762,738, filed Jun. 13, 2007 and WIPO Publication Number2007/147019, published Dec. 21, 2007. However, there is a need fortreatment of inflammatory disorders that would antagonize not onlyIL-17A and IL-23, but also IL-17F. The demonstrated in vivo activitiesof IL-23, IL-17A and IL-17F illustrate the clinical or therapeuticpotential of, and need for, antagonists of IL-23, IL-17A and IL-17F.Specifically, antibodies that bind to IL-23 and to IL-17A or IL-17F thatinhibit the immunological activities of both IL-17A and IL-17F wouldpossess such novel therapeutic qualities. The present invention servesthis need by providing antagonist molecules, including antibodies andantibody fragments that bind IL-23 and IL-17A or IL-17F, includingantagonists that are comprised on one molecule.

DETAILED DESCRIPTION OF THE INVENTION

The present invention addresses these needs by providing antagonists topro-inflammatory cytokines IL-17A, IL-17F, and IL-23. Antagonistsprovided by the invention are antibodies, or antibody fragments thatbind IL-17A and IL-17F, including antibodies that cross-react withIL-17A and IL-17F, and antibodies or antibody fragments that bind IL-23,including antibodies that bind the p19 subunit of IL-23. Thepolynucleotide sequence of the human IL-17A is shown in SEQ ID NO:1 andthe corresponding polypeptide sequence is shown in SEQ ID NO:2. Thepolynucleotide sequence of the human p19 subunit of IL-23 is shown inSEQ ID NO:3 and the corresponding polypeptide sequence is shown in SEQID NO:4. The polynucleotide sequence of the human IL-17F is shown in SEQID NO:5 and the corresponding polypeptide sequence is shown in SEQ IDNO:6.

IL-17A is a cytokine which stimulates the expression of IL-6, ICAM-1,IL-8, GM-CSF, and prostaglandin E2 expression, and plays a role in thepreferential maturation of CD34+ hematopoietic precursors intoneutrophils (Yao et al., J. Immunol. 155:5483 (1995); Fossiez et al., J.Exp. Med. 183:2593 (1996)).

The pro-inflammatory cytokines IL-17A and IL-17F have a high degree ofsequence similarity, share many biological properties, and are bothproduced by activated T cells. They have both been implicated as factorsthat contribute to the progression of various autoimmune andinflammatory diseases including multiple sclerosis, inflammatory boweldisease, rheumatoid arthritis and asthma. In fact, reagents that negateIL-17A function significantly ameliorate disease incidence and severityin several mouse models of human disease. IL-17A mediates its effectsthrough interaction with its cognate receptor, the IL-17 receptor(IL-17RA), and for IL-17F, IL-17RA. Thus, the present inventioncontemplates that a cross-reactive, also called herein cross-binding,antibody may be useful as an antagonist to both IL-17A and IL-17F, andthus to block both IL-17A and IL-17F. Accordingly, the present inventionaddresses this need by providing therapeutic molecules (e.g. antibodies)which may block, inhibit, reduce, antagonize or neutralize the activityof both IL-17A and IL-17F. Thus, the present invention is directed tobispecific antibodies, with one antibody portion comprising across-reactive antibody, or antibody fragment that binds IL-17A orIL-17F and one antibody portion comprising an antibody or antibodyfragment that binds the p19 subunit of IL-23, such as the antibodiesdescribed herein. The invention further provides uses therefor ininflammatory disease, as well as related compositions and methods.

IL-23 is a heterodimeric cytokine composed of a unique subunit, p19(herein referred to interchangeably as “IL-23”, “p19” and IL23/p19”),and the p40 subunit, which is shared with interleukin-12 (IL-12)(Oppmann, Immunity 13:715 (2000)). IL-23 has been found to stimulate theproduction and/or maintenance of IL-17 from activated CD4+ T cells inwhat has now been termed as a “new” T-helper (Th) subset, designatedTh17. A review of IL-23 cytokine and receptor biology is reviewed inHolscher, Curr. Opin. Invest. Drugs 6:489 (2005) and Langrish et al.Immunol Rev. 202:96 (2004). Similar to Th1 and Th2 lineages, Th17 cellshave most likely evolved to provide adaptive immunity to specificclasses of pathogens, such as extracellular bacteria. However,inappropriate Th17 responses have been strongly implicated in a growinglist of autoimmune disorders, including multiple sclerosis, rheumatoidarthritis, inflammatory bowel disease, and psoriasis.

In fact, both IL-17 and IL-23 have also been reported to play importantroles in many autoimmune diseases, such as multiple sclerosis,rheumatoid arthritis, Crohn's disease, and psoriasis. Both IL-23 andIL-17 are overexpressed in the central nervous system of humans withmultiple sclerosis and in mice undergoing an animal model of multiplesclerosis, experimental autoimmune encephalomyelitis (EAE). Theoverexpression is observed in mice when the EAE is induced by eithermyelin oligodendrocyte glycoprotein (MOG) 35-55 peptide- or proteolipidpeptide (PLP). Furthermore, neutralization of either IL-23/p19 or IL-17results in amelioration of EAE symptoms in mice (Park et al, Immunol.6:1133 (2005); Chen et al, J Clin Invest. 116:1317 (2006)).

It has also been demonstrated that IL-17 and Th17 cells can be producedfrom IL-23-independent sources, and the in vivo development of an IL-17effector response has been shown to be IL23-independent (Mangan et al,Nature 441:231 (2006)). Neutralization of IL-23 would theoreticallyeliminate existing IL-17 producing cells, but would not prevent thedevelopment of new Th17 cells.

Co-expression of IL-17F and IL-17A in HEK293 cells has been shown toresult in the production of the biologically active IL-17F/IL-17Aheterodimer, in addition to the IL-17F and IL-17A homodimers and thatactivated human CD4+ T cells produce the IL-17F/IL-17A heterodimer alongwith the corresponding homodimers. See for example, Wright, J. F. etal., J. Biol. Chem., Vol. 282, Issue 18: 13447-13455 2007.

The present invention concerns the inhibition of proinflammatorycytokines, IL-17A, IL-17F and IL-23/p19. This inhibition can be byadministration of one or more molecules that inhibit IL-17A or IL-17F,such as a cross-reactive antibody, and one or more molecules thatinhibit IL-23, such as an antibody or antibody fragment that binds thep19 subunit of IL-23. This inhibition can be by administration of onemolecule that comprises a binding entity that binds IL-17A or IL-17F andthat also binds the p19 subunit of IL-23. More specifically, the presentinvention concerns the inhibition or neutralization of IL-17A or IL-17Fand IL-23 (via p19) with a single antagonistic molecule or neutralizingentity. Since the portion of the single antagonistic molecule orneutralizing entity that binds IL-17A or IL-17F can bind either IL-17Aor IL-17F, administration of this molecule or entity will inhibit orneutralize IL-17A and IL-17F. As such, this portion of the molecule orentity will inhibit or neutralize IL-17A homodimers, IL-17F homodimers,and IL-17A/F heteterodimers. Thus, the single antagonistic molecule orneutralizing entity can be used to reduce, limit, neutralize, or blockthe proinflammatory effects of the IL-17A homodimer, the IL-17Fhomodimer, or the IL-17A/F heterodimer. Likewise, the singleantagonistic molecule or neutralizing entity can be used to reduce,limit, neutralize, or block the pro-cancerous effects of the IL-17Ahomodimer, the IL-17F homodimer, or the IL-17A/F heterodimer. In suchcases, the anti-IL-23p19 portion of the single antagonistic molecule orneutralizing entity is used to reduce, limit, neutralize, or blockproduction of new T cells that would produce IL-17A and/or IL-17F,including homodimers and heterodimers. The antagonistic molecules orneutralizing entities described herein can be used to treat autoimmunediseases, such as multiple sclerosis, inflammatory bowel disease, andpsoriasis. The antagonistic molecules or neutralizing entities describedherein can also be used to treat cancer, including angiogenesis.

The present invention is based on the surprising discovery thatantagonizing both IL-23 (via p19) and IL-17A is more effectivetherapeutically than neutralization of IL-23 alone (either via p19 orp40) or IL-17A alone and thus, necessary for the effective treatment ofinflammatory diseases (including cancers). See co-owned U.S. patentapplication Ser. No. 11/762,738, filed Jun. 13, 2007 and WIPOPublication Number 2007/147019, published Dec. 21, 2007.

The antagonistic molecule or neutralizing entity inhibits the activityof IL-17A or IL-17F and IL-23 (via the p19 subunit), and thus, inhibitsthe production, maintenance, and activity of new and existing IL-17A andIL-17F and IL-17-producing T cells (Th17). The invention furtherconcerns the use of antagonists or neutralizing entities of IL-17A,IL-17F, and IL-23/p19 in the treatment of inflammatory diseasescharacterized by the presence of elevated levels of IL-17A, IL-17F,and/or IL-23. The invention also concerns the use of antagonists toIL-17A, IL-17F, and IL-23/p19 in the treatment of cancers characterizedby the presence of elevated levels of IL-17A, IL-17F, and/or IL-23.

Accordingly, the present invention is directed to antagonizing IL-17A,IL-17F, and IL-23/p19. Antagonists, including antibodies and antibodyfragments of the present invention, which may block, inhibit, reduce,antagonize or neutralize the activity of IL-17A, IL-17F, (includinghomodimers and heterodimers), and IL-23/p19 will have advantages overtherapies that target only one of these three cytokines. The inventionfurther provides uses therefor in inflammatory disease and cancer, aswell as related compositions and methods.

One method to treat immune related diseases is to suppress the immuneresponse. Using the antagonists of the present invention (i.e.anti-IL-17A or anti-IL-17F and anti-IL-23/p19 antibodies) that inhibitmolecules having immune stimulatory activity would be beneficial in thetreatment of immune-mediated and inflammatory diseases. Molecules whichinhibit the immune response can be utilized (proteins directly or viathe use of antibody agonists) to inhibit the immune response and thusameliorate immune related disease.

IL-17, including IL-17A and IL-17F, has been identified as a potentcytokine that acts to induce proinflammatory responses in a wide varietyof peripheral tissues. IL-17 is a disulfide-linked homodimeric cytokineof about 32 kDa which is synthesized and secreted only by CD4+ activatedmemory T cells (reviewed in Fossiez et al., Int. Rev. Immunol., 16:541-551 [1998]). Specifically, IL-17 is synthesized as a precursorpolypeptide of 155 amino acids with an N-terminal signal sequence of19-23 residues and is secreted as a disulfide-linked homodimericglycoprotein. IL-17 is disclosed in WO9518826 (1995), WO9715320 (1997)and WO9704097 (1997), as well as U.S. Pat. No. 6,063,372.

Despite its restricted tissue distribution, IL-17 exhibits pleitropicbiological activities on various types of cells. IL-17 has been found tostimulate the production of many cytokines. It induces the secretion ofIL-6, IL-8, IL-12, leukemia inhibitory factor (LIF), prostaglandin E2,MCP-1 and G-CSF by adherent cells like fibroblasts, keratinocytes,epithelial and endothelial cells. IL-17 also has the ability to induceICAM-1 surface expression, proliferation of T cells, and growth anddifferentiation of CD34.sup.+ human progenitors into neutrophils. IL-17is also believed to play a key role in certain other autoimmunedisorders such as multiple sclerosis (Matusevicius et al., Mult. Scler.5:101 (1999); Park et al, Nat Immunol. 6:1133 (2005)). IL-17 has furtherbeen shown, by intracellular signalling, to stimulate Ca²⁺ influx and areduction in [cAMP], in human macrophages (Jovanovic et al, J. Immunol.160:3513 (1998)). Fibroblasts treated with IL-17 induce the activationof NF-kappa.B, (Yao et al., Immunity, 3:811 (1995), Jovanovic et al.,supra), while macrophages treated with it activate NF-kappa.B andmitogen-activated protein kinases (Shalom-Barek et al, J. Biol. Chem.273:27467 (1998)).

Despite its restricted tissue distribution, IL-17A exhibits pleitropicbiological activities on various types of cells. IL-17A has been foundto stimulate the production of many cytokines. It induces the secretionof IL-6, IL-8, IL-12, leukemia inhibitory factor (LIF), prostaglandinE2, MCP-1 and G-CSF by adherent cells like fibroblasts, keratinocytes,epithelial and endothelial cells. IL-17A also has the ability to induceICAM-1 surface expression, proliferation of T cells, and growth anddifferentiation of CD34⁺ human progenitors into neutrophils. IL-17A hasalso been implicated in bone metabolism, and has been suggested to playan important role in pathological conditions characterized by thepresence of activated T cells and TNF-.alpha. production such asrheumatoid arthritis and loosening of bone implants (Van Bezooijen etal., J. Bone Miner. Res. 14: 1513-1521 [1999]). Activated T cells ofsynovial tissue derived from rheumatoid arthritis patients were found tosecrete higher amounts of IL-17A than those derived from normalindividuals or osteoarthritis patients (Chabaud et al., Arthritis Rheum.42: 963-970 [1999]). It was suggested that this proinflammatory cytokineactively contributes to synovial inflammation in rheumatoid arthritis.Apart from its proinflammatory role, IL-17A seems to contribute to thepathology of rheumatoid arthritis by yet another mechanism. For example,IL-17A has been shown to induce the expression of osteoclastdifferentiation factor (ODF) mRNA in osteoblasts (Kotake et al., J.Clin. Invest., 103: 1345-1352 [1999]). ODF stimulates differentiation ofprogenitor cells into osteoclasts, the cells involved in boneresorption.

Since the level of IL-17A is significantly increased in synovial fluidof rheumatoid arthritis patients, it appears that IL-17A inducedosteoclast formation plays a crucial role in bone resorption inrheumatoid arthritis. IL-17A is also believed to play a key role incertain other autoimmune disorders such as multiple sclerosis(Matusevicius et al., Mult. Scler., 5: 101-104 [1999]). IL-17A hasfurther been shown, by intracellular signalling, to stimulate Ca.sup.2+influx and a reduction in [cAMP], in human macrophages (Jovanovic etal., J. Immunol., 160:3513 [1998]). Fibroblasts treated with IL-17Ainduce the activation of NF-.kappa.B, [Yao et al., Immunity, 3:811(1995), Jovanovic et al., supra], while macrophages treated with itactivate NF-.kappa.B and mitogen-activated protein kinases (Shalom-Bareket al., J. Biol. Chem., 273:27467 [1998]).

Additionally, IL-17A also shares sequence similarity with mammaliancytokine-like factor 7 that is involved in bone and cartilage growth.Other proteins with which IL-17A polypeptides share sequence similarityare human embryo-derived interleukin-related factor (EDIRF) andinterleukin-20.

The expression pattern of IL-17F appears to be similar to that ofIL-17A, such that it includes only activated CD4+ T cells and monocytes(Starnes et al. J. Immunol. 167: 4137-4140 [2001]). IL-17F has beendemonstrated to induce G-CSF, IL-6, and IL-8 in fibroblasts (Hymowitz etal, EMBO J. 20:5322-5341 [2001]) and TGF-b in endothelial cells (Starneset al. J. Immunol. 167: 4137-4140 [2001]). It has recently been reportedthat IL-23, a cytokine produced by dendritic cell, can mediate theproduction of both IL-17A and IL-17F, primarily in memory T cells(Aggarwal et al. J. Biol. Chem. 278:1910-1914 [2003]).

Moreover, over expression or upregulation of both IL-17A and IL-17F havebeen shown in arthritic and asthmatic individuals (reviewed in Moseleyet al. Cytokine Growth Factor Rev 14:155-174 [2003]). With regards toarthritis, these cytokines act in a manner characteristic to thecartilage and joint destruction that is associated with rheumatoid- andosteo-arthritis. For example, IL-17A and IL-17F have been demonstratedto enhance matrix degradation in articular cartilage explants viarelease of cartilage proteoglycan glycosaminoglycans and collagenfragments, while inhibiting the synthesis of new proteoglycans andcollagens (Cai et al. Cytokine 16:10-21 [2001]; Attur et al ArthritisRheum 44:2078-2083 [2001]).

Similar to IL-17A, overexpression of IL-17F in mice has also been shownto increase lung neutrophil recruitment and result in increasedexpression of Th1-associated cytokines in the lung, including IL-6,IFN-gamma, IP-10 and MIG (Starnes et al. J. Immunol. 167: 4137-4140[2001]). IL-17F was also upregulated in T cells from allergen-challengedasthmatics (Kawaguchi et al J. Immunol 167:4430-4435 [2001]), and foundto induce IL-6 and IL-8 production in NHBE. In contrast to IL-17A,IL-17F appears to inhibit angiogenesis in vitro (Starnes et al. J.Immunol. 167: 4137-4140 [2001]).

IL-17F mRNA was not detected by northern blot in various human tissuesbut was dramatically induced upon activation of CD4+ T cells andmonocytes. Id. In mice, Th2 cells and master cells were found to expressIL-17F upon activation. See Dumont, Expert Opin. Ther. Patents 13(3)(2003). Like IL-17A, the expression of IL-17F was also found to beupregulated by IL-23 in mouse.

The present invention also provides antibodies that bind both IL-17F andIL-23p19 and methods for using such antibodies. The antibodies may actas antagonists or agonists, and find utility for, among other things, invitro, in situ, or in vivo diagnosis or treatment of mammalian cells orpathological conditions associated with the presence (or absence) ofIL-17F and/or IL-23p19. In this embodiment, the antibody would bindIL-17F, but not IL-17A.

Antagonists to IL-17A, IL-17F, and IL-23 activity, such as theantagonists of the present invention (i.e. anti-IL-17A or anti-IL-17F oranti-IL-23/p19 antibodies), are useful in therapeutic treatment ofinflammatory diseases, particularly as antagonists to IL-17 A, IL-17F,and IL-23/p19, in the treatment of inflammatory diseases, particularlyin the treatment of multiple sclerosis, inflammatory bowel disease, andcancer. These antagonists are capable of binding, blocking, inhibiting,reducing, antagonizing or neutralizing IL-17A, IL-17F, their homodimersand heterodimers, and IL-23 (via p19) (either individually or together)in the treatment of atopic and contact dermatitis, multiple sclerosis,colitis, endotoxemia, arthritis, rheumatoid arthritis, psoriaticarthritis, adult respiratory disease (ARD), septic shock, multiple organfailure, inflammatory lung injury such as asthma, chronic obstructivepulmonary disease (COPD), airway hyper-responsiveness, chronicbronchitis, allergic asthma, psoriasis, eczema, IBS and inflammatorybowel disease (IBD) such as ulcerative colitis and Crohn's disease,Helicobacter pylori infection, intraabdominal adhesions and/or abscessesas results of peritoneal inflammation (i.e. from infection, injury,etc.), systemic lupus erythematosus (SLE), multiple sclerosis, systemicsclerosis, nephrotic syndrome, organ allograft rejection, graft vs. hostdisease (GVHD), kidney, lung, heart, etc. transplant rejection,streptococcal cell wall (SCW)-induced arthritis, osteoarthritis,gingivitis/periodontitis, herpetic stromal keratitis, restenosis,Kawasaki disease, and cancers/neoplastic diseases that are characterizedby IL-17 and/or IL-23 expression, including but not limited to prostate,renal, colon, ovarian and cervical cancer, and leukemias (Tartour et al,Cancer Res. 59:3698 (1999); Kato et al, Biochem. Biophys. Res. Commun.282:735 (2001); Steiner et al, Prostate. 56:171 (2003); Langowksi et al,Nature. May 10 [Epub ahead of print], (2006)).

The present invention provides novel antagonists of IL-17F and IL-23/p19and their uses in the treatment of inflammatory diseases and autoimmunediseases. The IL-17F and IL-23/p19 antagonists of the present invention,including the neutralizing anti-IL-17F and IL-23/p19 antibodies of thepresent invention, can be used to block, inhibit, reduce, antagonize orneutralize the activity of either IL-17F or IL-23 (via p19), or bothIL-17F and IL-23 (via p19) in the treatment of inflammation andinflammatory diseases such as multiple sclerosis, cancer (ascharacterized by the expression of IL-17F and/or IL-23), psoriasis,psoriatic arthritis, rheumatoid arthritis, endotoxemia, IBS, andinflammatory bowel disease (IBD), colitis, asthma, allograft rejection,immune mediated renal diseases, hepatobiliary diseases, atherosclerosis,promotion of tumor growth, or degenerative joint disease and otherinflammatory conditions disclosed herein.

The present invention provides isolated polypeptides that bind IL-17F(e.g., human IL-17F polypeptide sequence as shown in SEQ ID NO:6). Thepresent invention also provides isolated polypeptides as disclosed abovethat bind IL-23 (e.g., human IL-23 polypeptide sequence as shown in SEQID NO:4). More specifically, the present invention provides polypeptidesthat bind to the p19 subunit of IL-23 (e.g. human p19 polypeptidesequence as shown in SEQ ID NO:4).

The present invention also provides isolated polypeptides and epitopescomprising at least 15 contiguous amino acid residues of an amino acidsequence of SEQ ID NO:2 or 4. Illustrative polypeptides includepolypeptides that either comprise, or consist of SEQ ID NO:2 or 4, anantigenic epitope thereof. Moreover, the present invention also providesisolated polypeptides as disclosed above that bind to, block, inhibit,reduce, antagonize or neutralize the activity of IL-17F or IL-23.

Preferred embodiments of the invention include binding peptides,antibodies, and any fragments or permutations thereof that bind toIL-17F or IL-23/p19 (herein referred to interchangeably as “IL-17F/IL-23antagonists”, “IL-17F antagonists”, “IL-23 antagonists”, “p19antagonists” “IL-17F/IL-23 antibodies”, “IL-17F/p19 antibodies”, “IL-17Fantibodies”, “IL-23 antibodies”, “p19 antibodies” “IL-17F/IL-23antibodies”, “IL-17F/p19 antibodies”, “IL-17F/IL-23/p19 antibodies”etc.). Specifically, such binding peptides or antibodies are capable ofspecifically binding to both human IL-17F and IL-23 (via p19) and/or arecapable of modulating biological activities associated with either orboth IL-17F and IL-23, and thus are useful in the treatment of variousdiseases and pathological conditions such as inflammation andimmune-related diseases.

The invention include antibodies, and any fragments or permutationsthereof, that cross-reacts with IL-17A and IL-17F (herein refereed tointerchangeably as “cross-reactive antibodies”, “cross-bindingantibodies”, “A/F antibodies”, “IL-17A/F antibodies” etc.) as well asantibodies, including any fragments or permutations thereof, that bindIL-23p19. Specifically, such antibodies are capable of specificallybinding to both human IL-17A and IL-17F and/or are capable of modulatingbiological activities associated with either or both IL-17A and IL-17Fand/or their receptors, IL-17RA and IL-17RC, and thus are useful in thetreatment of various diseases and pathological conditions such as immunerelated diseases. Optionally, the antibody is a monoclonal antibody.

Thus, the present invention provides antibodies and antibody fragmentsthat specifically bind with IL-17 and/or IL-23 (via p19). Exemplaryantibodies include neutralizing antibodies, polyclonal antibodies,murine monoclonal antibodies, chimeric antibodies, humanized antibodiesderived from murine monoclonal antibodies, and human monoclonalantibodies. Illustrative antibody fragments including F(ab′)₂, F(ab)₂,Fab′, Fab, Fv, scFv, bispecific antibodies or antibody fragments, andminimal recognition units. Neutralizing antibodies preferably bindIL-17A, IL-17F, their homodimers or heterodimers, or IL-23/p19 such thatthe interaction of these ligands with their respective receptors isblocked, inhibited, reduced, antagonized or neutralized. That is, theneutralizing antibodies of the present invention can either bind, block,inhibit, reduce, antagonize or neutralize each of IL-17A, IL-17F, theirhomodimers or heterodimers, or IL-23 singly, or bind, block, inhibit,reduce, antagonize or neutralize IL-17A, IL-17F, their homodimers andheterodimers and IL-23 together. The present invention further includescompositions comprising a carrier and a peptide, polypeptide, orantibody described herein.

The present invention further includes pharmaceutical compositions,comprising a pharmaceutically acceptable carrier and a polypeptide orantibody described herein.

The present invention also provides fusion proteins, comprising anantagonist of the present invention and an immunoglobulin moiety. Insuch fusion proteins, the immunoglobulin moiety may be an immunoglobulinheavy chain constant region, such as a human Fc fragment. The presentinvention further includes isolated nucleic acid molecules that encodesuch fusion proteins. In another embodiment, the antibodies are linkedto one or more non-proteinaceous polymers selected from the groupconsisting of polyethylene glycol, polypropylene glycol, andpolyoxyalkylene, or to a cytotoxic agent or enzyme, or to aradioisotope, fluorescent compound or chemiluminescent compound.

In a particular embodiment, the present invention provides bispecificantibodies or binding proteins that bind both IL-17A, or IL-17F, andIL-23. Bispecific antibodies (BsAbs) are antibodies that have twodifferent antigen binding sites, such that the antibody specificallybinds to two different antigens. Antibodies having higher valencies(i.e., the ability to bind to more than two antigens) can also beprepared; they are referred to as multispecific antibodies.

The bispecific antibody can be a monoclonal antibody (MAb). Inparticular embodiments, the antibody is chimeric, or humanized, or fullyhuman. Fully human antibodies may be generated by procedures thatinvolve immunizing transgenic mice, wherein human immunoglobulin geneshave been introduced into the mice, as discussed below.

In yet other particular embodiments, there is provided the hybridomacell line which produces monoclonal antibodies of the present invention.In another embodiment, the IL-17/IL-23 antibodies are linked to one ormore non-proteinaceous polymers selected from the group consisting ofpolyethylene glycol, polypropylene glycol, and polyoxyalkylene, or to acytotoxic agent or enzyme, or to a radioisotope, fluorescent compound orchemiluminescent compound.

Compositions of the invention may include pharmaceutically acceptablecarriers or diluents. Preferably, the compositions will include one ormore antibodies in an amount which is therapeutically effective to treata pathological condition or disease.

Accordingly, antagonists of the present invention (i.e. antibodies orbinding peptides that bind IL-17A or IL-17F, and IL-23 either singly ortogether) are also useful to prepare medicines and medicaments for thetreatment of immune-related and inflammatory diseases, including forexample, systemic lupus erythematosis, arthritis, rheumatoid arthritis,osteoarthritis, demyelinating diseases of the central and peripheralnervous systems such as multiple sclerosis, idiopathic demyelinatingpolyneuropathy or Guillain-Barre syndrome, inflammatory bowel disease,colitis, ulcerative colitis, Crohn's disease, gluten-sensitiveenteropathy, cancer, neoplastic diseases, and angiogenesis. In aspecific aspect, such medicines and medicaments comprise atherapeutically effective amount of an anti-IL-17A/F cross-bindingantibody/IL-23 antibody with a pharmaceutically acceptable carrier. Inan embodiment, the admixture is sterile.

For example, the IL-17A/F cross-binding antibodies bind to an epitope onboth IL-17A and IL-17F, wherein said epitope comprises residues Ile(23),Lys (25), Gly(27), Thr (29) and Pro(34) of the following sequences ofhuman IL-17F and the equivalent sequence found in human IL-17A shownbelow. Residues 23, 25, 27, 29, and 34 are predicted to be on thesurface of both IL-17A and IL-17F and therefore are accessible to thebinding of an antibody of the present invention or an equivalent proteinbinding antagonist.

hIL17F (Ile23-Pro34 of SEQ ID NO: 6) IPKVGHTFFQKP hIL17A(Ile20-Pro31 of SEQ ID NO: 2) IVKAGITIPRNP

Optionally, the IL-17A/F antibodies bind to another epitope on bothIL-17A and IL-17F, wherein said epitope comprises residues Arg(67),Ser(68), Thr(69), Ser(70), Pro(71), Trp(72), Asn(73) of the followingsequences of human IL-17F and the equivalent sequence found in humanIL-17A, as shown below. Residues 69, 71 and 73 are predicted to be onthe surface of the bioactive cytokine and therefore are accessible tothe binding of an antibody of the present invention or equivalentprotein binding antagonist.

hIL17F (Arg67-Asn73 of SEQ ID NO: 6) RSTSPWN hIL17A(Arg69-Asn75 of SEQ ID NO: 2) RSTSPWN

Optionally, the IL-17A/F antibodies bind to another epitope on bothIL-17A and IL-17F, wherein said epitope comprises residues Asp(79),Pro(80), Asn(81), Arg(82), Tyr(83), Pro(84) and Ser(85) of the followingsequences of human IL-17F and the equivalent sequence found in humanIL-17A, as shown below. All residues of this epitope are predicted to beon the surface of the bioactive cytokine and therefore are accessible tothe binding of an antibody of the present invention or equivalentprotein binding antagonist.

hIL-17F (Asp79-Ser85 of SEQ ID NO: 6) DPNRYPS hIL-17A(Asp81-5er87 of SEQ ID NO: 2) DPERYPS

Optionally, the IL-17A/F antibodies bind to another epitope on bothIL-17A and IL-17F, wherein said epitope comprises residues Thr(146),Pro(147), Val(148), Ile(149), His(150), His(151), Val(152) of thefollowing sequences of human IL-17F and the corresponding sequence foundin human IL-17A, as shown below. These residues are predicted to be onthe surface of the bioactive cytokine and therefore to be accessible tothe binding of an antibody of the present invention or equivalentprotein binding antagonist.

hIL-17F (Thr146-Val152 of SEQ ID NO: 6) TPVIHHV hIL-17A(Thr148-Val154 of SEQ ID NO: 2) TPIVHHV

Optionally, the IL-17A/F antibodies bind to another epitope on bothIL-17A and IL-17F, wherein said epitope is a discontinuous epitopecomprising residues from two separate peptide chains of human IL-17F, asshown below; or the equivalent sequence found in human IL-17A, as shownbelow. Specifically, residues 105-109, 147-152 of hIL-17F and 107-111,148-154 of hIL-17A are predicted to be on the surface of the bioactivecytokine and therefore are accessible to the binding of an antibody ofthe present invention or equivalent protein binding antagonist.

hIL-17F Sequences (Asp105-Asn109 [DISMN] and Pro147-Val152 [PVIHHV] ofSEQ ID NO:6)

hIL-17A Sequences (Asp107-Asn111 [DYHMN] and Pro149-Val154 [PIVHHV] ofSEQ ID NO:2)

Optionally, the IL-17A/F antibodies bind to another epitope on bothIL-17A and IL-17F, wherein said epitope is a discontinuous epitopecomprising residues of two or three separate peptide chains of humanIL-17F, as shown below; or the equivalent sequence found in humanIL-17A. Specifically, residues 81, 82, 121, 132, 134 of hIL-17F and 83,84, 123, 134, 136 of hIL-17A are predicted to be on the surface of thebioactive cytokine and therefore to be accessible to the binding of anantibody of the present invention or equivalent protein bindingantagonist.

hIL-17F Sequences (Asp79-Ser85 [DPNRYPS] and Val119-Arg122 [VVRR] andSer130-Glu134 [SFQLE] of SEQ ID NO:6)

hIL-17A Sequences (Asp81-Ser87 [DPERYPS] and Val121-Arg124 [VLRR] andSer132-Glu136 [SFRLE] of SEQ ID NO:2)

Additionally, an epitope of IL-17A or to IL-17F to which neutralizingcross-reacting antibodies of the present invention may bind may be fromresidues 34 to residue 41 of SEQ ID NO: 2 (i.e., PNSEDKNF) or fromresidues 52 to 64 of SEQ ID NO: 2 (i.e., HNRNTNTNPKRSS). The an epitopeof IL-17A to which non-neutralizing antibodies of the present inventionmay bind may be from residues 77 to 85 of SEQ ID NO: 2 (i.e.,HRNEDPERY).

Likewise, epitopes of IL-23p19 to which antibodies of the presentinvention may bind may be from residues 55 to 66 of SEQ ID NO: 4 (i.e.,DLREEGDEETTN), from residues 74 to 85 (i.e., GDGCDPQGLRDN); fromresidues 137 to 146 (i.e., PEGHHWETQQ) and from residues 155 to 164(i.e, PWQRLLLRFK).

In a particular embodiment, the present invention provides bispecificantibodies with one binding entity that is cross-reactive for IL-17A andIL-17F and one binding entity that binds IL-23p19. Bispecific antibodies(BsAbs) are antibodies that have two different antigen binding sites,such that the antibody specifically binds to two different antigens.Antibodies having higher valencies (i.e., the ability to bind to morethan two antigens) can also be prepared; they are referred to asmultispecific antibodies.

In yet other particular embodiments, there is provided the hybridomacell line which produces monoclonal antibodies of the present invention.In another embodiment, the antibodies that bind to IL-17A or IL-17F arelinked to one or more non-proteinaceous polymers selected from the groupconsisting of polyethylene glycol, polypropylene glycol, andpolyoxyalkylene, or to a cytotoxic agent or enzyme, or to aradioisotope, fluorescent compound or chemiluminescent compound.

Typical methods of the invention include methods to treat pathologicalconditions or diseases in mammals associated with or resulting fromincreased or enhanced IL-17F expression and/or activity. In the methodsof treatment, IL-17F antibodies may be administered which preferablyblock or reduce the respective receptor binding or activation to theirreceptor(s).

The invention also provides compositions which comprise IL-17Fantibodies. Optionally, the compositions of the invention will includepharmaceutically acceptable carriers or diluents. Preferably, thecompositions will include one or more IL-17F antibodies in an amountwhich is therapeutically effective to treat a pathological condition ordisease.

As such, the present invention concerns compositions and methods usefulfor the diagnosis and treatment of immune related disease in mammals,including humans. The present invention is based on the identificationof antibodies that bind to IL-17F (including agonist and antagonistantibodies) which either stimulate or inhibit the immune response inmammals. Immune related diseases can be treated by suppressing orenhancing the immune response. Antibodies that enhance the immuneresponse stimulate or potentiate the immune response to an antigen.Antibodies which stimulate the immune response can be usedtherapeutically where enhancement of the immune response would bebeneficial. Alternatively, antibodies that suppress the immune responseattenuate or reduce the immune response to an antigen (e.g.,neutralizing antibodies) can be used therapeutically where attenuationof the immune response would be beneficial (e.g., inflammation).

Accordingly, antibodies that bind IL-17F of the present invention andare also useful to prepare medicines and medicaments for the treatmentof immune-related and inflammatory diseases, including for example,systemic lupus erythematosis, arthritis, psoriatic arthritis, rheumatoidarthritis, osteoarthritis, juvenile chronic arthritis,spondyloarthropathies, systemic sclerosis, idiopathic inflammatorymyopathies, Sjogren's syndrome, systemic vasculitis, sarcoidosis,autoimmune hemolytic anemia, autoimmune thrombocytopenia, thyroiditis,diabetes mellitus, immune-mediated renal disease, demyelinating diseasesof the central and peripheral nervous systems such as multiplesclerosis, idiopathic demyelinating polyneuropathy or Guillain-Barresyndrome, and chronic inflammatory demyelinating polyneuropathy,hepatobiliary diseases such as infectious, autoimmune chronic activehepatitis, primary biliary cirrhosis, granulomatous hepatitis, andsclerosing cholangitis, inflammatory bowel disease, colitis, Crohn'sdisease gluten-sensitive enteropathy, and endotoxemia, autoimmune orimmune-mediated skin diseases including bullous skin diseases, erythemamultiforme and atopic and contact dermatitis, psoriasis, neutrophilicdermatoses, cystic fibrosis, allergic diseases such as asthma, allergicrhinitis, food hypersensitivity and urticaria, cystic fibrosis,immunologic diseases of the lung such as eosinophilic pneumonia,idiopathic pulmonary fibrosis, adult respiratory disease (ARD), acuterespiratory distress syndrome (ARDS) and inflammatory lung injury suchas asthma, chronic obstructive pulmonary disease (COPD), airwayhyper-responsiveness, chronic bronchitis, allergic asthma andhypersensitivity pneumonitis, transplantation associated diseasesincluding graft and organ rejection and graft-versus-host-disease,septic shock, multiple organ failure, cancer and angiogenesis.

Models to test the effects of an antibody that binds IL-17A or IL-17Fand binds IL-23p19 on cancer are known in the art. One model is theRENCA model in which tumor growth is measured in groups of mice that areinjected s.c with the RENCA tumor on Day 0. Mice are then injected with50-200 ug control reagent or IL-17/IL-23 antagonist 1×-3×/week for 3weeks. Tumor volume is monitored 3×/week for 5 weeks. Significantlysmaller tumors compared to control reagent injected mice would suggestneutralization or inhibits tumor growth. Ten-week old female BALB/c mice(Charles River Laboratories) are injected s.c. on the right flank with0.1×106 RENCA cells on Day 0. Starting day 5, groups of mice(n=10/group) are injected i.p. with 50-200 ug of either control reagentor IL-17/IL-23 antagonist 1×-3×/week for 3 weeks. Tumor growth ismonitored 3×/week for 5 weeks using caliper measurements. Tumor volumeis calculated using the formula ½*(B)2*L (mm3).

Another model is the B16 Melanoma model. To test if an IL-17/IL-23antagonist has effects on tumor growth in mice, groups of mice areinjected s.c with the B16 tumor on Day 0. Mice are then injected with50-200 ug control reagent or IL-17/IL-23 antagonist 1×-3×/week for 3weeks. Tumor volume is monitored 3×/week for 5 weeks. Significantlysmaller tumors compared to control reagent injected mice would suggestneutralization or inhibits tumor growth. Ten-week old female C57Bl/6(Charles River Laboratories) are injected s.c. on the right flank with0.1×106 B16 cells on Day 0. Starting day 5, groups of mice (n=10/group)are injected i.p. with 50-200 ug of either control reagent orIL-17/IL-23 antagonist 1×-3×/week for 3 weeks Tumor growth is monitored3×/week for 5 weeks using caliper measurements. Tumor volume iscalculated using the formula ½*(B)2*L (mm3).

Another model is the LL/2 Lung Carcinoma model: to test if anIL-17/IL-23 antagonist has effects on tumor growth in mice, groups ofmice are injected s.c with the LL/2 tumor on Day 0. Mice are theninjected with 50-200 ug control reagent or IL-17/IL-23 antagonist1×-3×/week for 3 weeks. Tumor volume is monitored 3×/week for 5 weeks.Significantly smaller tumors compared to control reagent injected micewould suggest neutralization or inhibits tumor growth. Ten-week oldfemale C57BL/6 mice (Charles River Laboratories) are injected s.c. onthe right flank with 0.1×106 LL/2 cells on Day 0. Starting day 5, groupsof mice (n=10/group) are injected i.p. with 50-200 ug of either controlreagent or IL-17/IL-23 antagonist 1×-3×/week for 3 weeks Tumor growth ismonitored 3×/week for 5 weeks using caliper measurements. Tumor volumeis calculated using the formula ½*(B)2*L (mm3).

Another model measures tumor growth in the Ct-26 Colon Carcinoma model:to test if the IL-17/IL-23 antagonist has effects on tumor growth inmice, groups of mice are injected s.c with the CT-26 tumor on Day 0.Mice are then injected with 50-200 ug control reagent or IL-17/IL-23antagonist 1×-3×/week for 3 weeks. Tumor volume is monitored 3×/week for5 weeks. Significantly smaller tumors compared to control reagentinjected mice would suggest neutralization or inhibits tumor growth.Ten-week old female BALB/c mice (Charles River Laboratories) areinjected s.c. on the right flank with 0.1×106 CT-26 cells on Day 0.Starting day 5, groups of mice (n=10/group) are injected i.p. with50-200 ug of either control reagent or IL-17/IL-23 antagonist 1×-3×/weekfor 3 weeks Tumor growth is monitored 3×/week for 5 weeks using calipermeasurements. Tumor volume is calculated using the formula ½*(B)2*L(mm3).

Another model is the 4T1 Breast Carcinoma Model: to test if theIL-17/IL-23 antagonist has effects on tumor growth in mice, groups ofmice are injected s.c with the 4T1 tumor on Day 0. Mice are theninjected with 50-200 ug control reagent or IL-17/IL-23 antagonist1×-3×/week for 3 weeks. Tumor volume is monitored 3×/week for 5 weeks.Significantly smaller tumors compared to control reagent injected micewould suggest neutralization or inhibits tumor growth. Ten-week oldfemale BALB/c mice (Charles River Laboratories) are injected s.c. on theright flank with 0.1×106 4T1 cells on Day 0. Starting day 5, groups ofmice (n=10/group) are injected i.p. with 50-200 ug of either controlreagent or IL-17/IL-23 antagonist 1×-3×/week for 3 weeks Tumor growth ismonitored 3×/week for 5 weeks using caliper measurements. Tumor volumeis calculated using the formula ½*(B)2*L (mm3).

In a specific aspect, such medicines and medicaments comprise atherapeutically effective amount of an IL-17F/IL-23 antibody with apharmaceutically acceptable carrier. Preferably, the admixture issterile.

In one aspect, the present invention concerns an isolated antibody whichbinds to IL-17F. In another aspect, the antibody mimics the activity ofIL-17F (an agonist antibody) or conversely the antibody inhibits orneutralizes the activity of IL-17F (an antagonist antibody). In anotheraspect, the antibody is a monoclonal antibody, which preferably hasnonhuman complementarity determining region (CDR) residues and humanframework region (FR) residues.

In a further embodiment, the invention concerns a method of identifyingantagonist antibodies of IL-17F and IL-23/p19, said method comprisingcontacting both IL-17F and p19 with a candidate molecule and monitoringa biological activity mediated by IL-17F and/or IL-23. In anotherembodiment, the invention concerns a composition of matter comprising anIL-17F/IL-23 antagonist antibody which binds both IL-17F and IL-23 inadmixture with a carrier or excipient. In one aspect, the compositioncomprises a therapeutically effective amount of the IL-17F/IL-23antibody.

In an aspect, antagonistic IL-17A/F antibodies, are useful for: (a)decreasing infiltration of inflammatory cells into a tissue of a mammalin need thereof, (b) inhibiting or reducing an immune response in amammal in need thereof, (c) decreasing the activity of T-lymphocytes or(d) decreasing the proliferation of T-lymphocytes in a mammal in needthereof in response to an antigen.

In another aspect, the composition comprises a further activeingredient, which may, for example, be a further antibody or a cytotoxicor chemotherapeutic agent. Preferably, the composition is sterile. Inanother embodiment, the invention concerns a method of treating animmune related disorder in a mammal in need thereof, comprisingadministering to the mammal a therapeutically effective amount of anIL-17A/F-IL-23 antagonist.

In still another embodiment, the invention concerns an isolatedpolynucleotide that encodes a polypeptide of the present invention,wherein said polypeptide is capable of binding to IL-17A or IL-17F andIL-23p19. In an embodiment, the polypeptide inhibits the activity ofIL-17A or IL-17F and IL-23.

In still another embodiment, the invention concerns an isolatedpolypeptide of the present invention, wherein said polypeptide iscapable of binding to IL-17A or IL-17F and IL-23p19. In an embodiment,the polypeptide inhibits the activity of IL-17A or IL-17F and IL-23.

In yet another embodiment, the invention concerns a method forinhibiting IL-17 production by T cells comprising treating the T cellswith an antagonist of IL-23/p19 (IL-23).

Processes for producing the polypeptide of the invention are also hereindescribed, wherein those processes comprise culturing a host cellcomprising a vector which comprises the appropriate encoding nucleicacid molecule under conditions suitable for expression of said antibodyand recovering said antibody from the cell culture.

In a further embodiment, the invention concerns an article ofmanufacture, comprising: (a) a composition of matter comprising anantibody described herein; (b) a container containing said composition;and (c) a label affixed to said container, or a package insert includedin said container referring to the use of said antibody in the treatmentof an immune related disease.

The invention also provides articles of manufacture and kits whichinclude one or more antibodies described herein.

These and other aspects of the invention will become evident uponreference to the following detailed description. In addition, variousreferences are identified below and are incorporated by reference intheir entirety.

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules that lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.Thus, as used herein, the term “antibody” or “antibody peptide(s)”refers to an intact antibody, or a binding fragment thereof thatcompetes with the intact antibody for specific binding and includeschimeric, humanized, fully human, and bispecific antibodies. In certainembodiments, binding fragments are produced by recombinant DNAtechniques. In additional embodiments, binding fragments are produced byenzymatic or chemical cleavage of intact antibodies. Binding fragmentsinclude, but are not limited to, Fab, Fab′, F(ab)², F(ab′)², Fv, andsingle-chain antibodies.

The term “isolated antibody” as used herein refers to an antibody thathas been identified and separated and/or recovered from a component ofits natural environment. Contaminant components of its naturalenvironment are materials which would interfere with diagnostic ortherapeutic uses for the antibody, and may include enzymes, hormones,and other proteinaceous or nonproteinaceous solutes. In preferredembodiments, the antibody will be purified (1) to greater than 95% byweight of antibody as determined by the Lowry method, and mostpreferably more than 99% by weight, (2) to a degree sufficient to obtainat least 15 residues of N-terminal or internal amino acid sequence byuse of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGEunder reducing or nonreducing conditions using Coomassie blue or,preferably, silver stain. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

A “variant” antibody refers herein to a molecule which differs in aminoacid sequence from a “parent” antibody amino acid sequence by virtue ofaddition, deletion and/or substitution of one or more amino acidresidue(s) in the parent antibody sequence. In the preferred embodiment,the variant comprises one or more amino acid substitution(s) in one ormore hypervariable region(s) of the parent antibody. For example, thevariant may comprise at least one, e.g. from about one to about ten, andpreferably from about two to about five, substitutions in one or morehypervariable regions of the parent antibody. Ordinarily, the variantwill have an amino acid sequence having at least 75% amino acid sequenceidentity with the parent antibody heavy or light chain variable domainsequences, more preferably at least 80%, more preferably at least 85%,more preferably at least 90%, and most preferably at least 95%. Identityor homology with respect to this sequence is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical with the parent antibody residues, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity. None of N-terminal, C-terminal, or internalextensions, deletions, or insertions into the antibody sequence shall beconstrued as affecting sequence identity or homology. The variantretains the ability to bind human IL-17A or IL-lyF and IL-23 (via p19)and preferably has properties which are superior to those of the parentantibody. For example, the variant may have a stronger binding affinity,enhanced ability to inhibit IL-17A or IL-17F and IL-23-inducedinflammation. To analyze such properties, one should compare a Fab formof the variant to a Fab form of the parent antibody or a full lengthform of the variant to a full length form of the parent antibody, forexample. The variant antibody of particular interest herein is one whichdisplays at least about 10 fold, preferably at least about 20 fold, andmost preferably at least about 50 fold, enhancement in biologicalactivity when compared to the parent antibody.

The term “parent antibody” as used herein refers to an antibody which isencoded by an amino acid sequence used for the preparation of thevariant. Preferably, the parent antibody has a human framework regionand, if present, has human antibody constant region(s). For example, theparent antibody may be a humanized or human antibody.

The term “agonist” refers to any compound including a protein,polypeptide, peptide, antibody, antibody fragment, large molecule, orsmall molecule (less than 10 kD), that increases the activity,activation or function of another molecule.

The term “antagonist” refers to any compound including a protein,polypeptide, peptide, antibody, antibody fragment, large molecule, orsmall molecule (less than 10 kD), that decreases the activity,activation or function of another molecule.

The term “bind(ing) of a polypeptide” includes, but is not limited to,the binding of a ligand polypeptide of the present invention to areceptor; the binding of a receptor polypeptide of the present inventionto a ligand; the binding of an antibody of the present invention to anantigen or epitope; the binding of an antigen or epitope of the presentinvention to an antibody; the binding of an antibody of the presentinvention to an anti-idiotypic antibody; the binding of ananti-idiotypic antibody of the present invention to a ligand; the bidingof an anti-idiotypic antibody of the present invention to a receptor;the binding of an anti-anti-idiotypic antibody of the present inventionto a ligand, receptor or antibody, etc.

A “bispecific” or “bifunctional” antibody is a hybrid antibody havingtwo different heavy/light chain pairs and two different binding sites.Bispecific antibodies may be produced by a variety of methods including,but not limited to, fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai & Lachmann (1990), Clin. Exp. Immunol.79:315-321; Kostelny et al. (1992), J. Immunol. 148:1547-1553.

The term “chimeric antibody” or “chimeric antibodies” refers toantibodies whose light and heavy chain genes have been constructed,typically by genetic engineering, from immunoglobulin variable andconstant region genes belonging to different species. For example, thevariable segments of the genes from a mouse monoclonal antibody may bejoined to human constant segments, such as gamma 1 and gamma 3. Atypical therapeutic chimeric antibody is thus a hybrid protein composedof the variable or antigen-binding domain from a mouse antibody and theconstant domain from a human antibody, although other mammalian speciesmay be used. Specifically, a chimeric antibody is produced byrecombinant DNA technology in which all or part of the hinge andconstant regions of an immunoglobulin light chain, heavy chain, or both,have been substituted for the corresponding regions from anotheranimal's immunoglobulin light chain or heavy chain. In this way, theantigen-binding portion of the parent monoclonal antibody is graftedonto the backbone of another species' antibody. One approach, describedin EP 0239400 to Winter et al. describes the substitution of onespecies' complementarity determining regions (CDRs) for those of anotherspecies, such as substituting the CDRs from human heavy and light chainimmunoglobulin variable region domains with CDRs from mouse variableregion domains. These altered antibodies may subsequently be combinedwith human immunoglobulin constant regions to form antibodies that arehuman except for the substituted murine CDRs which are specific for theantigen. Methods for grafting CDR regions of antibodies may be found,for example in Riechmann et al. (1988) Nature 332:323-327 and Verhoeyenet al. (1988) Science 239:1534-1536.

The term “effective neutralizing titer” as used herein refers to theamount of antibody which corresponds to the amount present in the serumof animals (human or cotton rat) that has been shown to be eitherclinically efficacious (in humans) or to reduce virus by 99% in, forexample, cotton rats. The 99% reduction is defined by a specificchallenge of, e.g., 10³ pfu, 10⁴ pfu, 10⁵ pfu, 10⁶ pfu, 10⁷ pfu, 10⁸pfu, or 10⁹ pfu) of RSV.

As used herein, the term “epitope” refers to the portion of an antigento which an antibody specifically binds. Thus, the term “epitope”includes any protein determinant capable of specific binding to animmunoglobulin or T-cell receptor. Epitopic determinants usually consistof chemically active surface groupings of molecules such as amino acidsor sugar side chains and usually have specific three dimensionalstructural characteristics, as well as specific charge characteristics.More specifically, the term “IL-17 epitope”, “IL-23 epitope” and/or“IL-23/p19 epitope” as used herein refers to a portion of thecorresponding polypeptide having antigenic or immunogenic activity in ananimal, preferably in a mammal, and most preferably in a mouse or ahuman. An epitope having immunogenic activity is a portion of an IL-17Aor IL-17F or IL-23/p19 polypeptide that elicits an antibody response inan animal. An epitope having antigenic activity is a portion of anIL-17A or IL-17F or IL-23/p19 polypeptide to which an antibodyimmunospecifically binds as determined by any method well known in theart, for example, by immunoassays. Antigenic epitopes need notnecessarily be immunogenic. Such epitopes can be linear in nature or canbe a discontinuous epitope. Thus, as used herein, the term“conformational epitope” refers to a discontinuous epitope formed by aspatial relationship between amino acids of an antigen other than anunbroken series of amino acids.

The term “epitope tagged” when used herein refers to the anti-IL-17A oranti-Il-17F or anti-IL-23/p19 antibody fused to an “epitope tag”. Theepitope tag polypeptide has enough residues to provide an epitopeagainst which an antibody can be made, yet is short enough such that itdoes not interfere with activity of antibodies of the present invention.The epitope tag preferably is sufficiently unique so that the antibodythereagainst does not substantially cross-react with other epitopes.Suitable tag polypeptides generally have at least 6 amino acid residuesand usually between about 8-50 amino acid residues (preferably betweenabout 9-30 residues). Examples include the flu HA tag polypeptide andits antibody 12CA5 (Field et al. Mol. Cell. Biol. 8:2159-2165 (1988));the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto(Evan et al., Mol. Cell. Biol. 5(12):3610-3616(1985)); and the HerpesSimplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al.,Protein Engineering 3(6):547-553(1990)). In certain embodiments, theepitope tag is a “salvage receptor binding epitope”. As used herein, theterm “salvage receptor binding epitope” refers to an epitope of the Fcregion of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, or IgG₄) that isresponsible for increasing the in vivo serum half-life of the IgGmolecule.

The term “fragment” as used herein refers to a peptide or polypeptidecomprising an amino acid sequence of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least contiguous 80 amino acid residues, atleast contiguous 90 amino acid residues, at least contiguous 100 aminoacid residues, at least contiguous 125 amino acid residues, at least 150contiguous amino acid residues, at least contiguous 175 amino acidresidues, at least contiguous 200 amino acid residues, or at leastcontiguous 250 amino acid residues of the amino acid sequence of a IL-17or IL-23/p19 polypeptide or an antibody that immunospecifically binds toa either IL-17 or IL-23 (via p19) or both IL-17 and IL-23/p19polypeptide.

As used herein, the term “immunoglobulin” refers to a protein consistingof one or more polypeptides substantially encoded by immunoglobulingenes. One form of immunoglobulin constitutes the basic structural unitof an antibody. This form is a tetramer and consists of two identicalpairs of immunoglobulin chains, each pair having one light and one heavychain. In each pair, the light and heavy chain variable regions aretogether responsible for binding to an antigen, and the constant regionsare responsible for the antibody effector functions.

Full-length immunoglobulin “light chains” (about 25 Kd or 214 aminoacids) are encoded by a variable region gene at the NH2-terminus (about110 amino acids) and a kappa or lambda constant region gene at theCOOH-terminus. Full-length immunoglobulin “heavy chains” (about 50 Kd or446 amino acids), are similarly encoded by a variable region gene (about116 amino acids) and one of the other aforementioned constant regiongenes (about 330 amino acids). Heavy chains are classified as gamma, mu,alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM,IgA, IgD and IgE, respectively. Within light and heavy chains, thevariable and constant regions are joined by a “J” region of about 12 ormore amino acids, with the heavy chain also including a “D” region ofabout 10 more amino acids. (See generally, Fundamental Immunology (Paul,W., ed., 2nd ed. Raven Press, N.Y., 1989), Ch. 7 (incorporated byreference in its entirety for all purposes).

An immunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions. Thus, theterm “hypervariable region” refers to the amino acid residues of anantibody which are responsible for antigen binding. The hypervariableregion comprises amino acid residues from a “Complementarity DeterminingRegion” or “CDR” (Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)) and/or those residues from a “hypervariable loop”(Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-917) (both of which areincorporated herein by reference). “Framework Region” or “FR” residuesare those variable domain residues other than the hypervariable regionresidues as herein defined. The sequences of the framework regions ofdifferent light or heavy chains are relatively conserved within aspecies. Thus, a “human framework region” is a framework region that issubstantially identical (about 85% or more, usually 90-95% or more) tothe framework region of a naturally occurring human immunoglobulin. Theframework region of an antibody, that is the combined framework regionsof the constituent light and heavy chains, serves to position and alignthe CDR's. The CDR's are primarily responsible for binding to an epitopeof an antigen.

Accordingly, the term “humanized” immunoglobulin refers to animmunoglobulin comprising a human framework region and one or more CDR'sfrom a non-human (usually a mouse or rat) immunoglobulin. The non-humanimmunoglobulin providing the CDR's is called the “donor” and the humanimmunoglobulin providing the framework is called the “acceptor”.Constant regions need not be present, but if they are, they must besubstantially identical to human immunoglobulin constant regions, i.e.,at least about 85-90%, preferably about 95% or more identical. Hence,all parts of a humanized immunoglobulin, except possibly the CDR's, aresubstantially identical to corresponding parts of natural humanimmunoglobulin sequences. A “humanized antibody” is an antibodycomprising a humanized light chain and a humanized heavy chainimmunoglobulin. For example, a humanized antibody would not encompass atypical chimeric antibody as defined above, e.g., because the entirevariable region of a chimeric antibody is non-human.

As used herein, the term “human antibody” includes and antibody that hasan amino acid sequence of a human immunoglobulin and includes antibodiesisolated from human immunoglobulin libraries or from animals transgenicfor one or more human immunoglobulin and that do not express endogenousimmunoglobulins, as described, for example, by Kucherlapati et al. inU.S. Pat. No. 5,939,598.

The term “genetically altered antibodies” means antibodies wherein theamino acid sequence has been varied from that of a native antibody.Because of the relevance of recombinant DNA techniques in the generationof antibodies, one need not be confined to the sequences of amino acidsfound in natural antibodies; antibodies can be redesigned to obtaindesired characteristics. The possible variations are many and range fromthe changing of just one or a few amino acids to the complete redesignof, for example, the variable or constant region. Changes in theconstant region will, in general, be made in order to improve or altercharacteristics, such as complement fixation, interaction with membranesand other effector functions. Changes in the variable region will bemade in order to improve the antigen binding characteristics.

In addition to antibodies, immunoglobulins may exist in a variety ofother forms including, for example, single-chain or Fv, Fab, and(Fab′)₂, as well as diabodies, linear antibodies, multivalent ormultispecific hybrid antibodies (as described above and in detail in:Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and in singlechains (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85,5879-5883 (1988) and Bird et al., Science, 242, 423-426 (1988), whichare incorporated herein by reference). (See, generally, Hood et al.,“Immunology”, Benjamin, N.Y., 2nd ed. (1984), and Hunkapiller and Hood,Nature, 323, 15-16 (1986), which are incorporated herein by reference).

As used herein, the terms “single-chain Fv,” “single-chain antibodies,”“Fv” or “scFv” refer to antibody fragments that comprises the variableregions from both the heavy and light chains, but lacks the constantregions, but within a single polypeptide chain. Generally, asingle-chain antibody further comprises a polypeptide linker between theVH and VL domains which enables it to form the desired structure whichwould allow for antigen binding. Single chain antibodies are discussedin detail by Pluckthun in The Pharmacology of Monoclonal Antibodies,vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp.269-315 (1994). Various methods of generating single chain antibodiesare known, including those described in U.S. Pat. Nos. 4,694,778 and5,260,203; International Patent Application Publication No. WO 88/01649;Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra etal. (1988) Science 242:1038-1041, the disclosures of which areincorporated by reference for any purpose. In specific embodiments,single-chain antibodies can also be bi-specific and/or humanized.

A “Fab fragment” is comprised of one light chain and the C_(H1) andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule.

A “Fab′ fragment” contains one light chain and one heavy chain thatcontains more of the constant region, between the C_(H1) and C_(H2)domains, such that an interchain disulfide bond can be formed betweentwo heavy chains to form a F(ab′)₂ molecule.

A “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H1) andC_(H2) domains, such that an interchain disulfide bond is formed betweentwo heavy chains.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

The term “linear antibodies” refers to the antibodies described inZapata et al. Protein Eng. 8(10): 1057-1062 (1995). Briefly, theseantibodies comprise a pair of tandem Fd segments(V_(H)-C_(H1)-V_(H)-C_(H1)) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific.

The term “immunologically functional immunoglobulin fragment” as usedherein refers to a polypeptide fragment that contains at least thevariable domains of the immunoglobulin heavy and light chains. Animmunologically functional immunoglobulin fragment of the invention iscapable of binding to a ligand, preventing binding of the ligand to itsreceptor, interrupting the biological response resulting from ligandbinding to the receptor, or any combination thereof. Preferably, animmunologically functional immunoglobulin fragment of the inventionbinds specifically to either IL-17A or IL-17F and IL-23/p19.

The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. The term “monoclonalantibody” refers to an antibody that is derived from a single clone,including any eukaryotic, prokaryotic, or phage clone, and not themethod by which it is produced.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,α-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have alterations in sugarmoieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

The term “complement of a nucleic acid molecule” refers to a nucleicacid molecule having a complementary nucleotide sequence and reverseorientation as compared to a reference nucleotide sequence.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons as compared to areference nucleic acid molecule that encodes a polypeptide. Degeneratecodons contain different triplets of nucleotides, but encode the sameamino acid residue (i.e., GAU and GAC triplets each encode Asp).

The term “structural gene” refers to a nucleic acid molecule that istranscribed into messenger RNA (mRNA), which is then translated into asequence of amino acids characteristic of a specific polypeptide.

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a growth factor that has been separated from thegenomic DNA of a cell is an isolated DNA molecule. Another example of anisolated nucleic acid molecule is a chemically-synthesized nucleic acidmolecule that is not integrated in the genome of an organism. A nucleicacid molecule that has been isolated from a particular species issmaller than the complete DNA molecule of a chromosome from thatspecies.

A “nucleic acid molecule construct” is a nucleic acid molecule, eithersingle- or double-stranded, that has been modified through humanintervention to contain segments of nucleic acid combined and juxtaposedin an arrangement not existing in nature.

“Linear DNA” denotes non-circular DNA molecules having free 5′ and 3′ends. Linear DNA can be prepared from closed circular DNA molecules,such as plasmids, by enzymatic digestion or physical disruption.

“Complementary DNA (cDNA)” is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand. The term “cDNA” also refers to a clone of a cDNA moleculesynthesized from an RNA template.

A “promoter” is a nucleotide sequence that directs the transcription ofa structural gene. Typically, a promoter is located in the 5′ non-codingregion of a gene, proximal to the transcriptional start site of astructural gene. Sequence elements within promoters that function in theinitiation of transcription are often characterized by consensusnucleotide sequences. These promoter elements include RNA polymerasebinding sites, TATA sequences, CAAT sequences, differentiation-specificelements (DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993)), cyclicAMP response elements (CREs), serum response elements (SREs; Treisman,Seminars in Cancer Biol. 1:47 (1990)), glucocorticoid response elements(GREs), and binding sites for other transcription factors, such asCRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye etal., J. Biol. Chem. 269:25728 (1994)), SP1, cAMP response elementbinding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and octamerfactors (see, in general, Watson et al., eds., Molecular Biology of theGene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), andLemaigre and Rousseau, Biochem. J. 303:1 (1994)). If a promoter is aninducible promoter, then the rate of transcription increases in responseto an inducing agent. In contrast, the rate of transcription is notregulated by an inducing agent if the promoter is a constitutivepromoter. Repressible promoters are also known.

A “core promoter” contains essential nucleotide sequences for promoterfunction, including the TATA box and start of transcription. By thisdefinition, a core promoter may or may not have detectable activity inthe absence of specific sequences that may enhance the activity orconfer tissue specific activity.

A “regulatory element” is a nucleotide sequence that modulates theactivity of a core promoter. For example, a regulatory element maycontain a nucleotide sequence that binds with cellular factors enablingtranscription exclusively or preferentially in particular cells,tissues, or organelles. These types of regulatory elements are normallyassociated with genes that are expressed in a “cell-specific,”“tissue-specific,” or “organelle-specific” manner.

An “enhancer” is a type of regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

“Heterologous DNA” refers to a DNA molecule, or a population of DNAmolecules, that does not exist naturally within a given host cell. DNAmolecules heterologous to a particular host cell may contain DNA derivedfrom the host cell species (i.e., endogenous DNA) so long as that hostDNA is combined with non-host DNA (i.e., exogenous DNA). For example, aDNA molecule containing a non-host DNA segment encoding a polypeptideoperably linked to a host DNA segment comprising a transcriptionpromoter is considered to be a heterologous DNA molecule. Conversely, aheterologous DNA molecule can comprise an endogenous gene operablylinked with an exogenous promoter. As another illustration, a DNAmolecule comprising a gene derived from a wild-type cell is consideredto be heterologous DNA if that DNA molecule is introduced into a mutantcell that lacks the wild-type gene.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides.”

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

A peptide or polypeptide encoded by a non-host DNA molecule is a“heterologous” peptide or polypeptide.

A “cloning vector” is a nucleic acid molecule, such as a plasmid,cosmid, or bacteriophage, that has the capability of replicatingautonomously in a host cell. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites that allowinsertion of a nucleic acid molecule in a determinable fashion withoutloss of an essential biological function of the vector, as well asnucleotide sequences encoding a marker gene that is suitable for use inthe identification and selection of cells transformed with the cloningvector. Marker genes typically include genes that provide tetracyclineresistance or ampicillin resistance.

An “expression vector” is a nucleic acid molecule encoding a gene thatis expressed in a host cell. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and such agene is said to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter.

A “recombinant host” is a cell that contains a heterologous nucleic acidmolecule, such as a cloning vector or expression vector. In the presentcontext, an example of a recombinant host is a cell that produces anantagonist of the present invention from an expression vector. Incontrast, such an antagonist can be produced by a cell that is a“natural source” of said antagonist, and that lacks an expressionvector.

A “fusion protein” is a hybrid protein expressed by a nucleic acidmolecule comprising nucleotide sequences of at least two genes. Forexample, a fusion protein can comprise at least part of a IL-17RApolypeptide fused with a polypeptide that binds an affinity matrix. Sucha fusion protein provides a means to isolate large quantities of IL-17RAusing affinity chromatography.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule termed a “ligand.” This interaction mediates theeffect of the ligand on the cell. Receptors can be membrane bound,cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormonereceptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor,growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor,erythropoietin receptor and IL-6 receptor). Membrane-bound receptors arecharacterized by a multi-domain structure comprising an extracellularligand-binding domain and an intracellular effector domain that istypically involved in signal transduction. In certain membrane-boundreceptors, the extracellular ligand-binding domain and the intracellulareffector domain are located in separate polypeptides that comprise thecomplete functional receptor.

In general, the binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell, which in turnleads to an alteration in the metabolism of the cell. Metabolic eventsthat are often linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids.

The term “secretory signal sequence” denotes a DNA sequence that encodesa peptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

An “isolated polypeptide” is a polypeptide that is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, or otherproteinaceous impurities associated with the polypeptide in nature.Typically, a preparation of isolated polypeptide contains thepolypeptide in a highly purified form, i.e., at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure,such as 96%, 97%, or 98% or more pure, or greater than 99% pure. One wayto show that a particular protein preparation contains an isolatedpolypeptide is by the appearance of a single band following sodiumdodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the proteinpreparation and Coomassie Brilliant Blue staining of the gel. However,the term “isolated” does not exclude the presence of the samepolypeptide in alternative physical forms, such as dimers oralternatively glycosylated or derivatized forms.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “expression” refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, lymphotoxins, co-stimulatory molecules, hematopoieticfactors, and the like, and synthetic analogs of these molecules.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity ofless than 10⁹ M⁻¹.

As used herein, a “therapeutic agent” is a molecule or atom which isconjugated to an antibody moiety to produce a conjugate which is usefulfor therapy. Examples of therapeutic agents include drugs, toxins,immunomodulators, chelators, boron compounds, photoactive agents ordyes, and radioisotopes.

A “detectable label” is a molecule or atom which can be conjugated to anantibody moiety to produce a molecule useful for diagnosis. Examples ofdetectable labels include chelators, photoactive agents, radioisotopes,fluorescent agents, paramagnetic ions, or other marker moieties.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075 (1985);Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione Stransferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)),substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2:95 (1991). DNA molecules encoding affinity tags areavailable from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.).

A “target polypeptide” or a “target peptide” is an amino acid sequencethat comprises at least one epitope, and that is expressed on a targetcell, such as a tumor cell, or a cell that carries an infectious agentantigen. T cells recognize peptide epitopes presented by a majorhistocompatibility complex molecule to a target polypeptide or targetpeptide and typically lyse the target cell or recruit other immune cellsto the site of the target cell, thereby killing the target cell.

Due to the imprecision of standard analytical methods, molecular weightsand lengths of polymers are understood to be approximate values. Whensuch a value is expressed as “about” X or “approximately” X, the statedvalue of X will be understood to be accurate to +10%.

The antibodies of the invention comprise a first antibody portion thatbinds to IL-17A or IL-17F and second antibody portion that binds toIL-23 (via p19). In some embodiments, the antibodies of the inventionspecifically bind a monomeric form of both IL-17A or IL-17F. In someembodiments, the antibodies of the invention bind a homodimeric form ofeither IL-17A or IL-17F. In some embodiments, the antibodies of theinvention bind a heterodimeric form of IL-17A/F. In still otherembodiments, the antibodies of the invention specifically bind amultimeric form of IL-17 (e.g., a heterodimeric form). For instance,IL-17 can form a heterodimer with any other member of the IL-17 familyof ligands, such as IL-17B, IL-17C, or IL-17F. In some embodiments, theantibodies of the invention bind a homodimeric or heterodimeric form ofIL-23 (via binding to the p19 subunit) Preferred antibodies of theinvention block the biological activities of IL-17A or IL-17F and IL-23,either singly or together.

The antibodies of the invention include portions of intact antibodiesthat retain antigen-binding specificity, for example, Fab fragments,Fab′ fragments, F(ab′)₂ fragments, F(v) fragments, heavy chain monomersor dimers, light chain monomers or dimers, dimers consisting of oneheavy and one light chain, and the like. Thus, antigen bindingfragments, as well as full-length dimeric or trimeric polypeptidesderived from the above-described antibodies are themselves useful.

The direct use of rodent monoclonal antibodies (MAbs) as humantherapeutic agents led to human anti-rodent antibody (“HARA”) (forexample, human anti-mouse antibody (“HAMA”)) responses which occurred ina significant number of patients treated with the rodent-derivedantibody (Khazaeli, et al., (1994) Immunother. 15:42-52). Chimericantibodies containing fewer murine amino acid sequences are believed tocircumvent the problem of eliciting an immune response in humans.

Refinement of antibodies to avoid the problem of HARA responses led tothe development of “humanized antibodies.” Humanized antibodies areproduced by recombinant DNA technology, in which at least one of theamino acids of a human immunoglobulin light or heavy chain that is notrequired for antigen binding has been substituted for the correspondingamino acid from a nonhuman mammalian immunoglobulin light or heavychain. For example, if the immunoglobulin is a mouse monoclonalantibody, at least one amino acid that is not required for antigenbinding is substituted using the amino acid that is present on acorresponding human antibody in that position. Without wishing to bebound by any particular theory of operation, it is believed that the“humanization” of the monoclonal antibody inhibits human immunologicalreactivity against the foreign immunoglobulin molecule.

As a non-limiting example, a method of performing complementaritydetermining region (CDR) grafting may be performed by sequencing themouse heavy and light chains of the antibody of interest that binds tothe target antigen (e.g., IL-17 and/or IL-23/p19) and geneticallyengineering the CDR DNA sequences and imposing these amino acidsequences to corresponding human V regions by site directed mutagenesis.Human constant region gene segments of the desired isotype are added,and the “humanized” heavy and light chain genes are co-expressed inmammalian cells to produce soluble humanized antibody. A typicalexpression cell is a Chinese Hamster Ovary (CHO) cell. Suitable methodsfor creating the chimeric antibodies may be found, for example, in Joneset al. (1986) Nature 321:522-525; Riechmann (1988) Nature 332:323-327;Queen et al. (1989) Proc. Nat. Acad. Sci. USA 86:10029; and Orlandi etal. (1989) Proc. Natl. Acad. Sci. USA 86:3833.

Queen et al. (1989) Proc. Nat. Acad. Sci. USA 86:10029-10033 and WO90/07861 describe the preparation of a humanized antibody. Human andmouse variable framework regions were chosen for optimal proteinsequence homology. The tertiary structure of the murine variable regionwas computer-modeled and superimposed on the homologous human frameworkto show optimal interaction of amino acid residues with the mouse CDRs.This led to the development of antibodies with improved binding affinityfor antigen (which is typically decreased upon making CDR-graftedchimeric antibodies). Alternative approaches to making humanizedantibodies are known in the art and are described, for example, inTempest (1991) Biotechnology 9:266-271.

The antibodies of the invention may be used alone or as immunoconjugateswith a cytotoxic agent. In some embodiments, the agent is achemotherapeutic agent. In some embodiments, the agent is aradioisotope, including, but not limited to Lead-212, Bismuth-212,Astatine-211, Iodine-131, Scandium-47, Rhenium-186, Rhenium-188,Yttrium-90, Iodine-123, Iodine-125, Bromine-77, Indium-111, andfissionable nuclides such as Boron-10 or an Actinide. In otherembodiments, the agent is a toxin or cytotoxic drug, including but notlimited to ricin, modified Pseudomonas enterotoxin A, calicheamicin,adriamycin, 5-fluorouracil, and the like. Methods of conjugation ofantibodies and antibody fragments to such agents are known in theliterature.

The antibodies of the invention include derivatives that are modified,e.g., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody from bindingto its epitope. Examples of suitable derivatives include, but are notlimited to fucosylated antibodies and fragments, glycosylated antibodiesand fragments, acetylated antibodies and fragments, pegylated antibodiesand fragments, phosphorylated antibodies and fragments, and amidatedantibodies and fragments. The antibodies and derivatives thereof of theinvention may themselves by derivatized by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherproteins, and the like. In some embodiments of the invention, at leastone heavy chain of the antibody is fucosylated. In some embodiments, thefucosylation is N-linked. In some preferred embodiments, at least oneheavy chain of the antibody comprises a fucosylated, N-linkedoligosaccharide.

The antibodies of the invention include variants having single ormultiple amino acid substitutions, deletions, additions, or replacementsthat retain the biological properties (e.g., block the binding of IL-17Aor IL-17F and/or IL-23 to their respective receptors, block thebiological activity of IL-17A or IL-17F and IL-23, binding affinity) ofthe antibodies of the invention. The skilled person can produce variantshaving single or multiple amino acid substitutions, deletions, additionsor replacements. These variants may include, inter alia: (a) variants inwhich one or more amino acid residues are substituted with conservativeor nonconservative amino acids, (b) variants in which one or more aminoacids are added to or deleted from the polypeptide, (c) variants inwhich one or more amino acids include a substituent group, and (d)variants in which the polypeptide is fused with another peptide orpolypeptide such as a fusion partner, a protein tag or other chemicalmoiety, that may confer useful properties to the polypeptide, such as,for example, an epitope for an antibody, a polyhistidine sequence, abiotin moiety and the like. Antibodies of the invention may includevariants in which amino acid residues from one species are substitutedfor the corresponding residue in another species, either at theconserved or nonconserved positions. In another embodiment, amino acidresidues at nonconserved positions are substituted with conservative ornonconservative residues. The techniques for obtaining these variants,including genetic (suppressions, deletions, mutations, etc.), chemical,and enzymatic techniques, are known to the person having ordinary skillin the art. Antibodies of the invention also include antibody fragments.A “fragment” refers to polypeptide sequences which are preferably atleast about 40, more preferably at least to about 50, more preferably atleast about 60, more preferably at least about 70, more preferably atleast about 80, more preferably at least about 90, and more preferablyat least about 100 amino acids in length, and which retain somebiological activity or immunological activity of the full-lengthsequence, for example, the ability to block the binding of IL-17 and/orIL-23 to their respective receptors, block the biological activity ofIL-17 and IL-23, binding affinity.

The invention also encompasses fully human antibodies such as thosederived from peripheral blood mononuclear cells of ovarian, breast,renal, colorectal, lung, endometrial, or brain cancer patients. Suchcells may be fused with myeloma cells, for example, to form hybridomacells producing fully human antibodies against both IL-17A or IL-17F andIL-23/p19.

The invention also encompasses bispecific antibodies that bind to bothIL-17A or IL-17F and IL-23 (via p19).

The antibodies of the invention are preferably nontoxic as demonstrated,for example, in in vivo toxicology studies.

The antibodies and derivatives thereof of the invention have bindingaffinities that include a dissociation constant (K_(d)) of less than1×10⁻². In some embodiments, the K_(d) is less than 1×10⁻³. In otherembodiments, the K_(d) is less than 1×10⁻⁴. In some embodiments, theK_(d) is less than 1×10⁻⁵. In still other embodiments, the K_(d) is lessthan 1×10⁻⁶. In other embodiments, the K_(d) is less than 1×10⁻⁷. Inother embodiments, the K_(d) is less than 1×10⁻⁸. In other embodiments,the v is less than 1×10⁻⁹. In other embodiments, the v is less than1×10⁻¹⁰. In still other embodiments, the K_(d) is less than 1×10⁻¹¹. Insome embodiments, the K_(d) is less than 1×10⁻¹². In other embodiments,the K_(d) is less than 1×10⁻¹³. In other embodiments, the K_(d) is lessthan 1×10⁻¹⁴. In still other embodiments, the K_(d) is less than1×10⁻¹⁵.

The invention also includes nucleic acids encoding the heavy chainand/or light chain of the antibodies of the invention. Nucleic acids ofthe invention include nucleic acids having at least 80%, more preferablyat least about 90%, more preferably at least about 95%, and mostpreferably at least about 98% homology to nucleic acids of theinvention. The terms “percent similarity”, “percent identity” and“percent homology” when referring to a particular sequence are used asset forth in the University of Wisconsin GCG software program. Nucleicacids of the invention also include complementary nucleic acids. In someinstances, the sequences will be fully complementary (no mismatches)when aligned. In other instances, there may be up to about a 20%mismatch in the sequences. In some embodiments of the invention areprovided nucleic acids encoding both a heavy chain and a light chain ofan antibody of the invention.

Nucleic acids of the invention can be cloned into a vector, such as aplasmid, cosmid, bacmid, phage, artificial chromosome (BAC, YAC) orvirus, into which another genetic sequence or element (either DNA orRNA) may be inserted so as to bring about the replication of theattached sequence or element. In some embodiments, the expression vectorcontains a constitutively active promoter segment (such as but notlimited to CMV, SV40, Elongation Factor or LTR sequences) or aninducible promoter sequence such as the steroid inducible pIND vector(Invitrogen), where the expression of the nucleic acid can be regulated.Expression vectors of the invention may further comprise regulatorysequences, for example, an internal ribosomal entry site. The expressionvector can be introduced into a cell by transfection, for example.

The invention also provides methods of producing antibodies, includingmonoclonal antibodies that specifically bind to IL-17A or IL-17F andIL-23p19, either singly or together. Antibodies of the invention may beproduced in vivo or in vitro. Standard methods are known for creatingmonoclonal antibodies including, but are not limited to, the hybridomatechnique (see Kohler & Milstein, (1975) Nature 256:495-497); the triomatechnique; the human B-cell hybridoma technique (see Kozbor et al.(1983) Immunol. Today 4:72) and the EBV hybridoma technique to producehuman monoclonal antibodies (see Cole, et al. in MONOCLONAL ANTIBODIESAND CANCER THERAPY, Alan R. Liss, Inc., 1985, pp. 77-96).

Both IL-17F and IL-23 may be purified from cells or from recombinantsystems using a variety of well-known techniques for isolating andpurifying proteins. For example, but not by way of limitation, bothIL-17F and IL-23 may be isolated based on the apparent molecular weightof the protein by running the protein on an SDS-PAGE gel and blottingthe proteins onto a membrane. Thereafter, the appropriate size bandcorresponding to either protein may be cut from the membrane and used asan immunogen in animals directly, or by first extracting or eluting theprotein from the membrane. As an alternative example, the protein may beisolated by size-exclusion chromatography alone or in combination withother means of isolation and purification.

The invention also provides methods of producing monoclonal antibodiesthat specifically bind to homodimeric, heterodimeric, and/or multimericforms of both IL-17F and IL-23/p19. These different forms may bepurified from cells or from recombinant systems using a variety ofwell-known techniques for isolating and purifying proteins. For example,but not by way of limitation, both IL-17F and IL-23/p19 may be isolatedbased on the apparent molecular weight of the protein by running theprotein on an SDS-PAGE gel and blotting the proteins onto a membrane.Thereafter, the appropriate size band corresponding to each may be cutfrom the membrane and used as an immunogen in animals directly, or byfirst extracting or eluting the protein from the membrane. As analternative example, the protein may be isolated by size-exclusionchromatography alone or in combination with other means of isolation andpurification.

Other means of purification are available in such standard referencetexts as Zola, Monoclonal Antibodies: Preparation And Use Of MonoclonalAntibodies And Engineered antibody Derivatives (Basics: From BackgroundTo Bench) Springer-Verlag Ltd., New York, 2000; Basic Methods InAntibody Production And Characterization, Chapter 11, “AntibodyPurification Methods,” Howard and Bethell, Eds., CRC Press, 2000;Antibody Engineering (Springer Lab Manual.), Kontermann and Dubel, Eds.,Springer-Verlag, 2001.

For in vivo antibody production, animals are generally immunized witheither IL-17A, IL-17F, or IL-23 or an immunogenic portion of either. Theantigen is generally combined with an adjuvant to promoteimmunogenicity. Adjuvants vary according to the species used forimmunization. Examples of adjuvants include, but are not limited to:Freund's complete adjuvant (“FCA”), Freund's incomplete adjuvant(“FIA”), mineral gels (e.g., aluminum hydroxide), surface activesubstances (e.g., lysolecithin, pluronic polyols, polyanions), peptides,oil emulsions, keyhole limpet hemocyanin (“KLH”), dinitrophenol (“DNP”),and potentially useful human adjuvants such as Bacille Calmette-Guerin(“BCG”) and Corynebacterium parvum. Such adjuvants are also well knownin the art. Immunization may be accomplished using well-knownprocedures. The dose and immunization regimen will depend on the speciesof mammal immunized, its immune status, body weight, and/or calculatedsurface area, etc. Typically, blood serum is sampled from the immunizedmammals and assayed for anti-IL-17 and IL-23/p19 antibodies usingappropriate screening assays as described below, for example.

A common method for producing humanized antibodies is to graft CDRsequences from a MAb (produced by immunizing a rodent host) onto a humanIg backbone, and transfection of the chimeric genes into Chinese HamsterOvary (CHO) cells which in turn produce a functional Ab that is secretedby the CHO cells (Shields, R. L., et al. (1995) Anti-IgE monoclonalantibodies that inhibit allergen-specific histamine release. Int Arch.Allergy Immunol. 107:412-413). The methods described within thisapplication are also useful for generating genetic alterations within Iggenes or chimeric Igs transfected within host cells such as rodent celllines, plants, yeast and prokaryotes (Frigerio L, et al. (2000)Assembly, secretion, and vacuolar delivery of a hybrid immunoglobulin inplants. Plant Physiol. 123:1483-1494).

Splenocytes from immunized animals may be immortalized by fusing thesplenocytes (containing the antibody-producing B cells) with an immortalcell line such as a myeloma line. Typically, myeloma cell line is fromthe same species as the splenocyte donor. In one embodiment, theimmortal cell line is sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). In someembodiments, the myeloma cells are negative for Epstein-Barr virus (EBV)infection. In preferred embodiments, the myeloma cells areHAT-sensitive, EBV negative and Ig expression negative. Any suitablemyeloma may be used. Murine hybridomas may be generated using mousemyeloma cell lines (e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O-Ag14 myeloma lines). These murine myeloma lines are available fromthe ATCC. These myeloma cells are fused to the donor splenocytespolyethylene glycol (“PEG”), preferably 1500 molecular weightpolyethylene glycol (“PEG 1500”). Hybridoma cells resulting from thefusion are selected in HAT medium which kills unfused and unproductivelyfused myeloma cells. Unfused splenocytes die over a short period of timein culture. In some embodiments, the myeloma cells do not expressimmunoglobulin genes.

Hybridomas producing a desired antibody which are detected by screeningassays such as those described below may be used to produce antibodiesin culture or in animals. For example, the hybridoma cells may becultured in a nutrient medium under conditions and for a time sufficientto allow the hybridoma cells to secrete the monoclonal antibodies intothe culture medium. These techniques and culture media are well known bythose skilled in the art. Alternatively, the hybridoma cells may beinjected into the peritoneum of an unimmunized animal. The cellsproliferate in the peritoneal cavity and secrete the antibody, whichaccumulates as ascites fluid. The ascites fluid may be withdrawn fromthe peritoneal cavity with a syringe as a rich source of the monoclonalantibody.

Hybridomas expressing mouse monoclonal antibodies that cross bind tohuman IL-17A and human IL-17F were produced using methods similar tothose described above were deposited with the American Type TissueCulture Collection (ATCC; 10801 University Blvd, Manassas Va.20110-2209) patent depository as original deposits under the BudapestTreaty and were given the following ATCC Accession Nos.: clone339.15.5.3 (ATCC Patent Deposit Designation PTA-7987, deposited on Nov.7, 2006); clone 339.15.3.6 (ATCC Patent Deposit Designation PTA-7988,deposited on Nov. 7, 2006); and clone 339.15.6.16 (ATCC Patent DepositDesignation PTA-7989, deposited on Nov. 7, 2006. These monoclonalantibodies and hybridomas are further described in co-owned US PatentPublication No. 2007-0218065, published Sep. 20, 2007 and in U.S. patentapplication Ser. No. 11/741,189, filed Apr. 27, 2007, hereinincorporated by reference.

Another non-limiting method for producing human antibodies is describedin U.S. Pat. No. 5,789,650 which describes transgenic mammals thatproduce antibodies of another species (e.g., humans) with their ownendogenous immunoglobulin genes being inactivated. The genes for theheterologous antibodies are encoded by human immunoglobulin genes. Thetransgenes containing the unrearranged immunoglobulin encoding regionsare introduced into a non-human animal. The resulting transgenic animalsare capable of functionally rearranging the transgenic immunoglobulinsequences and producing a repertoire of antibodies of various isotypesencoded by human immunoglobulin genes. The B-cells from the transgenicanimals are subsequently immortalized by any of a variety of methods,including fusion with an immortalizing cell line (e.g., a myeloma cell).

The antibodies of the present invention may also be prepared in vitrousing a variety of techniques known in the art. For example, but not byway of limitation, fully human monoclonal antibodies against IL-17A orIL-17F and IL-23/p19 may be prepared by using in vitro-primed humansplenocytes (Boerner et al. (1991) J. Immunol. 147:86-95).

Alternatively, for example, the antibodies of the invention may beprepared by “repertoire cloning” (Persson et al. (1991) Proc. Nat. AcadSci. USA 88:2432-2436; and Huang and Stollar (1991) J. Immunol. Methods141:227-236). Further, U.S. Pat. No. 5,798,230 describes preparation ofhuman monoclonal antibodies from human B antibody-producing B cells thatare immortalized by infection with an Epstein-Barr virus that expressesEpstein-Barr virus nuclear antigen 2 (EBNA2). EBNA2, required forimmortalization, is then inactivated resulting in increased antibodytiters.

In another embodiment, antibodies of the invention are formed by invitro immunization of peripheral blood mononuclear cells (“PBMCs”). Thismay be accomplished by any means known in the art, such as, for example,using methods described in the literature (Zafiropoulos et al. (1997) J.Immunological Methods 200:181-190).

Within an aspect the invention provides a method for inhibitinginflammation in a mammal comprising administering an antagonist of IL-23and an antagonist of IL-17A or IL-17F to the mammal, wherein theantagonist of IL-17A or IL-17F can bind IL-17A or IL-17F. Within anembodiment, the antagonist of IL-17A or IL-17F is an antibody orantibody fragment. Within another embodiment, the antibody or antibodyfragment is a cross-reactive antibody to IL-17A and IL-17F. Withinanother embodiment the antagonist of IL-23 binds the p19 subunit. Withinan embodiment the antagonist of IL-23 is an antibody or antibodyfragment.

The invention provides a method for inhibiting inflammation in a mammalcomprising administering an antagonist of IL2-3 and an antagonist ofIL-17A or IL-17F, wherein the antagonist of IL-17A or IL-17F comprisesan antibody or antibody fragment comprising a HCDR1 amino acid sequence,a HCDR2 amino acid sequence, and a HCDR3 amino acid sequence of thevariable heavy region selected from the group consisting of: thevariable heavy region of the hybridoma of ATCC Patent DepositDesignation PTA-7987; the variable heavy region of the hybridoma of ATCCPatent Deposit Designation PTA-7988; and the variable heavy region ofthe hybridoma of ATCC Patent Deposit Designation PTA-7989 and whereinthe variable light region comprises a LCDR1 amino acid sequence, a LCDR2amino acid sequence, and a LCDR3 amino acid sequence of the variablelight region selected from the group consisting of: the variable lightregion of the hybridoma of ATCC Patent Deposit Designation PTA-7987; thevariable light region of the hybridoma of ATCC Patent DepositDesignation PTA-7988; and the variable light region of the hybridoma ofATCC Patent Deposit Designation PTA-7989. Within an embodiment theantibody or antibody fragment that binds IL-17A or IL-17F comprises anamino acid sequence of the variable heavy region selected from the groupconsisting of: a) the variable heavy region of the hybridoma of ATCCPatent Deposit Designation PTA-7987; the variable heavy region of thehybridoma of ATCC Patent Deposit Designation PTA-7988; and the variableheavy region of the hybridoma of ATCC Patent Deposit DesignationPTA-7989. Within a different embodiment, the antibody or antibodyfragment that binds IL-17A or IL-17F comprises an amino acid sequence ofthe variable light region selected from the group consisting of: thevariable light region of the hybridoma of ATCC Patent DepositDesignation PTA-7987; the variable light region of the hybridoma of ATCCPatent Deposit Designation PTA-7988; and the variable light region ofthe hybridoma of ATCC Patent Deposit Designation PTA-7989. Within anembodiment the antagonist of IL2-3 and the antagonist of IL-17A orIL-17F are contained on one molecule.

The invention provides a method for inhibiting inflammation in a mammalcomprising administering an antagonist of IL2-3 and an antagonist ofIL-17A or IL-17F, wherein the antagonist of IL-17A or IL-17F comprisesan antibody or antibody fragment comprising an amino acid sequence ofthe variable heavy region and an amino acid sequence of the variablelight region and wherein the amino acid sequence of the variable heavyregion is selected from the group consisting of: the amino acid sequenceof the variable heavy region of the hybridoma of ATCC Patent DepositDesignation PTA-7987; the amino acid sequence of the variable heavyregion of the hybridoma of ATCC Patent Deposit Designation PTA-7988; andthe amino acid sequence of the variable heavy region of the hybridoma ofATCC Patent Deposit Designation PTA-7989 and wherein the variable lightregion comprises the amino acid sequence of the variable light regionselected from the group consisting of: the amino acid sequence of thevariable light region of the hybridoma of ATCC Patent DepositDesignation PTA-7987; the amino acid sequence of the variable lightregion of the hybridoma of ATCC Patent Deposit Designation PTA-7988; andthe amino acid sequence of the variable light region of the hybridoma ofATCC Patent Deposit Designation PTA-7989. Within an embodiment theantagonist of IL2-3 and the antagonist of IL-17A or IL-17F are containedon one molecule.

Within aspects, the antibody or antibody fragment is humanized. Withinother aspects the antibody is chimeric. Within other aspects theantibody fragment is selected from the group consisting of Fv, Fab,Fab′, F(ab)2, and F(ab′)2.

The invention provides a method for inhibiting inflammation in a mammalcomprising administering an antagonist of IL2-3 and an antagonist ofIL-17A or IL-17F, wherein the antagonist of IL-23 comprises an antibodyor antibody fragment that binds the p19 subunit of IL-23 and wherein theantibody or antibody fragment comprises a HCDR1 amino acid sequence, aHCDR2 amino acid sequence, and a HCDR3 amino acid sequence of the aminoacid sequence of variable heavy region as shown in SEQ ID NO: 8, SEQ IDNO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28,SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO:38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ IDNO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66,SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO:76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ IDNO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO:104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO:122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO:140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQID NO: 186, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO:193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO:211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO:229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO:247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 265, SEQ ID NO:267, and SEQ ID NO: 268 and wherein the variable light region comprisesa LCDR1 amino acid sequence, a LCDR2 amino acid sequence, and a LCDR3amino acid sequence of the amino acid sequence of variable light regionas shown in SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23,SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO:33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ IDNO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61,SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO:71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ IDNO: 81, SEQ ID NO: 83, SEQ ID NO:85, SEQ ID NO: 87, SEQ ID NO: 89, SEQID NO: 91, SEQ ID NO: 93, SEQ ID NO:95, SEQ ID NO: 97, SEQ ID NO: 99,SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ IDNO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117,SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ IDNO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135,SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ IDNO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153,SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ IDNO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171,SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ IDNO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 188, SEQ ID NO: 190,SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ IDNO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208,SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ IDNO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226,SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ IDNO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244,SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ IDNO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262,SEQ ID NO: 263, SEQ ID NO: 264, or SEQ ID NO: 266. Within an embodimentthe antagonist of IL2-3 and the antagonist of IL-17A or IL-17F arecontained on one molecule. Within an embodiment the variable heavyregion of the antibody or antibody fragment that binds the p19 subunitof IL-23 comprises an amino acid sequence as shown in SEQ ID NO: 8, SEQID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18,SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO:28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ IDNO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56,SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ IDNO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94,SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO:104, SEQ ID NO: 106v 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO:114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO:132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:186, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO:203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO:221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO:239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO:257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 265, SEQ ID NO: 267, andSEQ ID NO: 268. Within an embodiment the variable light region of theantibody or antibody fragment that binds the p19 subunit of IL-23comprises an amino acid sequence as shown in SEQ ID NO:7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ IDNO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47,SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO:57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ IDNO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO:85,SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ IDNO:95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO:113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO:131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO:149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO:185, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO:204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO:258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, orSEQ ID NO: 266. Within an embodiment the antagonist of IL2-3 and theantagonist of IL-17A or IL-17F are contained on one molecule.

The invention provides a method of inhibiting inflammation comprisingadministering an antagonist to a mammal wherein the antagonist is anantibody or antibody fragment that binds the p19 subunit of IL-23comprises a variable heavy region and a variable light region andwherein the amino acid sequence of the variable heavy region is shown inSEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ IDNO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44,SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO:54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ IDNO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82,SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO:92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ IDNO: 102, SEQ ID NO: 104, SEQ ID NO: 106v 108, SEQ ID NO: 110, SEQ ID NO:112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO:148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:184, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO:201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO:219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO:237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO:255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 265, SEQID NO: 267, and SEQ ID NO: 268 and wherein the amino acid sequence ofthe variable light region is shown in SEQ ID NO:7, SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29,SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ IDNO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67,SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO:77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO:85, SEQ IDNO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO:95, SEQID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO:105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO:123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO:141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO:159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO:177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO:196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQID NO: 260, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, or SEQ IDNO: 266. Within an embodiment the antagonist of IL2-3 and the antagonistof IL-17A or IL-17F are contained on one molecule.

The invention provides a method for inhibiting inflammation in a mammalcomprising administering an antagonist of IL-23 and an antagonist ofIL-17A or IL-17F to the mammal, wherein the antagonist of IL-17A orIL-17F can bind IL-17A or IL-17F, and wherein the inflammation isassociated with a disease selected from the group consisting of multiplesclerosis (MS), chronic inflammation, autoimmune diabetes, rheumatoidarthritis (RA) and other arthritic conditions, asthma, systhemic lupuserythrematosus, psoriasis, Crohn's Disease, ulcerative colitis,irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD).

The invention provides an antibody or antibody fragment that is a singlechain antibody. The invention provides an antibody or antibody fragmentthat is a bispecific antibody. The invention provides an antibody thatis a tascFv, a biscFv, or a BiAb antibody.

The invention provides a method for inhibiting inflammation in a mammalcomprising administering an antagonist of IL-23 and an antagonist ofIL-17A or IL-17F to the mammal, wherein the antagonist of IL-17A orIL-17F can bind IL-17A or IL-17F, and wherein the antibody or antibodyfragment binds an IL-17F epitope, wherein said epitope is selected fromthe group consisting of: a) an epitope comprising amino acid residues23, 25, 27, 29 and 34 of SEQ ID NO:6; b) an epitope comprising aminoacid residues 23-34 of SEQ ID NO:6; c) an epitope comprising amino acidresidues 67-73 of SEQ ID NO:6; d) an epitope comprising amino acidresidues 79-85 of SEQ ID NO:6; e) an epitope comprising amino acidresidues 146-152 of SEQ ID NO:4; f) an epitope comprising at least oneamino acid residue from residues 105-109 and at least one amino acidresidue from residues 147-152 of SEQ ID NO:6; g) an epitope comprisingat least one amino acid residue from residues 79-85, and at least oneamino acid residue from residues 119-122 and at least one amino acidresidue from residues 130-134 of SEQ ID NO:6; h) an epitope comprisingamino acid residues 34 to 41 of SEQ ID NO: 6; i) an epitope comprisingamino acid residues 52 to 64 of SEQ ID NO: 6; and j) an epitopecomprising amino acid residues 77 to 85 of SEQ ID NO: 6.

The invention provides a method for inhibiting inflammation in a mammalcomprising administering an antagonist of IL-23 and an antagonist ofIL-17A or IL-17F to the mammal, wherein the antagonist of IL-17A orIL-17F can bind IL-17A or IL-17F, and wherein the antibody or antibodyfragment binds an IL-17A epitope, wherein said epitope is selected fromthe group consisting of: a) an epitope comprising amino acid residues23, 25, 27, 29 and 34 of SEQ ID NO:2; b) an epitope comprising aminoacid residues 20-31 of SEQ ID NO:2; c) an epitope comprising amino acidresidues 69-75 of SEQ ID NO:2; d) an epitope comprising amino acidresidues 81-87 of SEQ ID NO:2; e) an epitope comprising amino acidresidues 148-154 of SEQ ID NO:2; f) an epitope comprising at least oneamino acid residue from residues 107-111 and at least one amino acidresidue from residues 149-154 of SEQ ID NO:2; g) an epitope comprisingat least one amino acid residue from residues 81-87 and at least oneamino acid residue from residues 121-124 and at least one amino acidresidue from residues 132-136 of SEQ ID NO:2; h) an epitope comprisingamino acid residues 34 to 41 of SEQ ID NO: 2; i) an epitope comprisingamino acid residues 52 to 64 of SEQ ID NO: 2; and j) an epitopecomprising amino acid residues 77 to 85 of SEQ ID NO: 2.

The invention provides a method for inhibiting inflammation in a mammalcomprising administering an antagonist of IL-23 and an antagonist ofIL-17A or IL-17F to the mammal, wherein the antagonist of IL-17A orIL-17F can bind IL-17A or IL-17F, and wherein the antibody or antibodyfragment binds an IL-1723 epitope, wherein said epitope is selected fromthe group consisting of: a) an epitope comprising amino acid residues 55to 66 of SEQ ID NO: 4; b) an epitope comprising amino acid residues 74to 85 of SEQ ID NO: 4; and c) an epitope comprising amino acid residues155 to 164 of SEQ ID NO: 4.

Within an aspect the antibody or antibody fragment also comprises a PEGmoiety. Within an aspect the antibody or antibody fragment alsocomprises an Fc moiety. Within an aspect the antibody or antibodyfragment is bivalent, trivalent, or tetravalent.

The invention provides a method for treating disease characterized byelevated expression of IL-17A, IL-17F or IL-23 in a mammalian subject,comprising administering to said subject an effective amount of anantagonist of IL-17A, IL-17F and IL-23.

The invention provides an antagonist of IL-23 and of IL-17A or IL-17Fcomprising a an antibody or antibody fragment that binds the p19 subunitof IL-23 as shown in SEQ ID NO: 4 and that comprises an antibody orantibody fragment that binds IL-17A as shown in SEQ ID NO: 2 or thatbinds IL-17F as shown in SEQ ID NO: 6. Within an embodiment the antibodyor antibody fragment that binds IL-17A is a cross-reactive antibody thatbinds IL-17F.

The invention provides an antagonist of IL-23 and of IL-17A or IL-17Fcomprising an antibody or antibody fragment that binds IL-17A or IL-17Fwherein the antibody or antibody fragment comprises a HCDR1 amino acidsequence, a HCDR2 amino acid sequence, and a HCDR3 amino acid sequenceof the variable heavy region selected from the group consisting of: a)the variable heavy region of the hybridoma of ATCC Patent DepositDesignation PTA-7987; b) the variable heavy region of the hybridoma ofATCC Patent Deposit Designation PTA-7988; and c) the variable heavyregion of the hybridoma of ATCC Patent Deposit Designation PTA-7989 andwherein the variable light region comprises a LCDR1 amino acid sequence,a LCDR2 amino acid sequence, and a LCDR3 amino acid sequence of thevariable light region selected from the group consisting of: d) thevariable light region of the hybridoma of ATCC Patent DepositDesignation PTA-7987; e) the variable light region of the hybridoma ofATCC Patent Deposit Designation PTA-7988; and f) the variable lightregion of the hybridoma of ATCC Patent Deposit Designation PTA-7989.Within an embodiment the antibody or antibody fragment that binds IL-17Aor IL-17F comprises an amino acid sequence of the variable heavy regionselected from the group consisting of: a. the variable heavy region ofthe hybridoma of ATCC Patent Deposit Designation PTA-7987; b. thevariable heavy region of the hybridoma of ATCC Patent DepositDesignation PTA-7988; and c. the variable heavy region of the hybridomaof ATCC Patent Deposit Designation PTA-7989. Within another embodiment,the antibody or antibody fragment that binds IL-17A or IL-17F comprisesan amino acid sequence of the variable light region selected from thegroup consisting of: a. the variable light region of the hybridoma ofATCC Patent Deposit Designation PTA-7987; b. the variable light regionof the hybridoma of ATCC Patent Deposit Designation PTA-7988; and c. thevariable light region of the hybridoma of ATCC Patent DepositDesignation PTA-7989. Within an embodiment the antagonist of IL2-3 andthe antagonist of IL-17A or IL-17F are contained on one molecule.

The invention provides an antagonist of IL-23 and of IL-17A or IL-17F,wherein the antibody or antibody fragment that binds IL-17A or IL-17Fcomprises an amino acid sequence of the variable heavy region and anamino acid sequence of the variable light region and wherein the aminoacid sequence of the variable heavy region is selected from the groupconsisting of: the amino acid sequence of the variable heavy region ofthe hybridoma of ATCC Patent Deposit Designation PTA-7987; the aminoacid sequence of the variable heavy region of the hybridoma of ATCCPatent Deposit Designation PTA-7988; and the amino acid sequence of thevariable heavy region of the hybridoma of ATCC Patent DepositDesignation PTA-7989 and wherein the variable light region comprises theamino acid sequence of the variable light region selected from the groupconsisting of: the amino acid sequence of the variable light region ofthe hybridoma of ATCC Patent Deposit Designation PTA-7987; the aminoacid sequence of the variable light region of the hybridoma of ATCCPatent Deposit Designation PTA-7988; and the amino acid sequence of thevariable light region of the hybridoma of ATCC Patent DepositDesignation PTA-7989. Within an embodiment the antagonist of IL2-3 andthe antagonist of IL-17A or IL-17F are contained on one molecule.

Within an aspect the antibody or antibody fragment is humanized. Withinan aspect the antibody or antibody fragment is chimeric. Within anaspect the antibody fragment is selected from the group consisting ofFv, Fab, Fab′, F(ab)2, and F(ab′)2.

The invention provides an antagonist of IL-23 and of IL-17A or IL-17F,wherein the antagonist comprises an antibody or antibody fragment thatbinds the p19 subunit of IL-23 and wherein the antibody or antibodyfragment comprises a HCDR1 amino acid sequence, a HCDR2 amino acidsequence, and a HCDR3 amino acid sequence of the amino acid sequence ofvariable heavy region as shown in SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30,SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO:40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ IDNO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68,SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO:78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ IDNO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO:106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO:124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO:195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO:213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO:231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO:249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQID NO: 259, SEQ ID NO: 261, SEQ ID NO: 265, SEQ ID NO: 267, and SEQ IDNO: 268 and wherein the variable light region comprises a LCDR1 aminoacid sequence, a LCDR2 amino acid sequence, and a LCDR3 amino acidsequence of the amino acid sequence of variable light region as shown inSEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ IDNO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53,SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO:63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ IDNO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQID NO: 83, SEQ ID NO:85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91,SEQ ID NO: 93, SEQ ID NO:95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO:101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO:119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO:137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO:155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQID NO: 183, SEQ ID NO: 185, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO:228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO:263, SEQ ID NO: 264, or SEQ ID NO: 266. Within an embodiment theantagonist of IL2-3 and the antagonist of IL-17A or IL-17F are containedon one molecule.

The invention provides an antagonist of IL-23 and of IL-17A or IL-17F,wherein the antagonist comprises an antibody or antibody fragment thatbinds the p19 subunit of IL-23 and wherein the antibody or antibodyfragment comprises an amino acid sequence of a variable heavy region asshown in SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO:34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ IDNO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO:72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ IDNO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100,SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106v 108, SEQ ID NO: 110, SEQID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQID NO: 184, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO:191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO:209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO:227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO:245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO:265, SEQ ID NO: 267, and SEQ ID NO: 268. Within an embodiment theantagonist of IL2-3 and the antagonist of IL-17A or IL-17F are containedon one molecule.

The invention provides an antagonist of IL-23 and of IL-17A or IL-17F,wherein the antagonist comprises an antibody or antibody fragment thatbinds the p19 subunit of IL-23 wherein the antibody or antibody fragmentcomprises an amino acid sequence of the variable light region as shownin SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ IDNO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43,SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO:53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ IDNO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81,SEQ ID NO: 83, SEQ ID NO:85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO:91, SEQ ID NO: 93, SEQ ID NO:95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ IDNO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109,SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ IDNO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127,SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ IDNO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145,SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ IDNO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163,SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ IDNO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181,SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 188, SEQ ID NO: 190, SEQ IDNO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200,SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ IDNO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218,SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ IDNO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236,SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ IDNO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254,SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ IDNO: 263, SEQ ID NO: 264, or SEQ ID NO: 266. Within an embodiment theantagonist of IL2-3 and the antagonist of IL-17A or IL-17F are containedon one molecule.

The invention provides an antagonist of IL-23 and of IL-17A or IL-17F,wherein the antagonist comprises an antibody or antibody fragment thatbinds the p19 subunit of IL-23 and comprises an amino acid sequence of avariable heavy region and an amino acid sequence of a variable lightregion and wherein the amino acid sequence of the variable heavy regionis shown in SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14,SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ IDNO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52,SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO:62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ IDNO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90,SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO:100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106v 108, SEQ ID NO:110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO:164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 189, SEQID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO:199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO:217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO:235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO:253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQID NO: 265, and SEQ ID NO: SEQ ID NO: 267, and SEQ ID NO: 268 andwherein the amino acid sequence of the variable light region is shown inSEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ IDNO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53,SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO:63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ IDNO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQID NO: 83, SEQ ID NO:85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91,SEQ ID NO: 93, SEQ ID NO:95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO:101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO:119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO:137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO:155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQID NO: 183, SEQ ID NO: 185, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO:228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO:263, SEQ ID NO: 264, or SEQ ID NO: 266. Within an embodiment theantagonist of IL2-3 and the antagonist of IL-17A or IL-17F are containedon one molecule.

The invention provides an antagonist of IL-23 and of IL-17A or IL-17F,wherein antagonist can be used to treat inflammation is associated witha disease selected from the group consisting of multiple sclerosis (MS),chronic inflammation, autoimmune diabetes, rheumatoid arthritis (RA) andother arthritic conditions, asthma, systhemic lupus erythrematosus,psoriasis, Crohn's Disease, ulcerative colitis, irritable bowel syndrome(IBS) and inflammatory bowel disease (IBD).

The invention provides an antagonist of IL-23 and of IL-17A or IL-17F,wherein the antagonist comprises an antibody or antibody fragment thatbinds an IL-17F epitope, wherein said epitope is selected from the groupconsisting of: a) an epitope comprising amino acid residues 23, 25, 27,29 and 34 of SEQ ID NO:6; b) an epitope comprising g amino acid residues23-34 of SEQ ID NO:6; c) an epitope comprising amino acid residues 67-73of SEQ ID NO:6; d) an epitope comprising amino acid residues 79-85 ofSEQ ID NO:6; e) an epitope comprising amino acid residues 146-152 of SEQID NO:6; f) an epitope comprising at least one amino acid residue fromresidues 105-109 and at least one amino acid residue from residues147-152 of SEQ ID NO:6; g) an epitope comprising at least one amino acidresidue from residues 79-85, and at least one amino acid residue fromresidues 119-122 and at least one amino acid residue from residues130-134 of SEQ ID NO:6; h) an epitope comprising amino acid residues 34to 41 of SEQ ID NO: 6; i) an epitope comprising amino acid residues 52to 64 of SEQ ID NO: 6; and j) an epitope comprising amino acid residues77 to 85 of SEQ ID NO: 6.

The invention provides an antagonist of IL-23 and of IL-17A or IL-17F,wherein the antagonist comprises an antibody or antibody fragment thatbinds an IL-17A epitope, wherein said epitope is selected from the groupconsisting of: a) an epitope comprising amino acid residues 23, 25, 27,29 and 34 of SEQ ID NO:2; b) an epitope comprising amino acid residues20-31 of SEQ ID NO:2; c) an epitope comprising amino acid residues 69-75of SEQ ID NO:2; d) an epitope comprising amino acid residues 81-87 ofSEQ ID NO:2; e) an epitope comprising amino acid residues 148-154 of SEQID NO:2; f) an epitope comprising at least one amino acid residue fromresidues 107-111 and at least one amino acid residue from residues149-154 of SEQ ID NO:2; g) an epitope comprising at least one amino acidresidue from residues 81-87 and at least one amino acid residue fromresidues 121-124 and at least one amino acid residue from residues132-136 of SEQ ID NO:2; h) an epitope comprising amino acid residues 34to 41 of SEQ ID NO: 2; i) an epitope comprising amino acid residues 52to 64 of SEQ ID NO: 2; and j) an epitope comprising amino acid residues77 to 85 of SEQ ID NO: 2.

The invention provides an antagonist of IL-23 and of IL-17A or IL-17F,wherein the antagonist comprises an antibody or antibody fragment thatbinds an IL-23p19 epitope selected from the group consisting of: a) anepitope comprising amino acid residues 55 to 66 of SEQ ID NO: 4; b) anepitope comprising amino acid residues 74 to 85 of SEQ ID NO: 4; and c)an epitope comprising amino acid residues 155 to 164 of SEQ ID NO: 4.

The invention provides an antagonist of IL-23 and of IL-17A or IL-17F,wherein the antagonist comprises an antibody or antibody fragmentcomprises a PEG moiety. The invention provides an antagonist of IL-23and of IL-17A or IL-17F, wherein the antagonist comprises an Fc moiety.The invention provides an antagonist of IL-23 and of IL-17A or IL-17F,wherein the antagonist is bivalent, trivalent, or tetravalent.

In a specific embodiment, bispecific and single chain antibodies thatbind both IL-17A or IL-17F and IL-23 are made. One method comprisesfusing hybridoma cells that secrete a monoclonal antibody that crossbinds with IL-17A and IL-17F, with hybridoma cells that secrete amonoclonal antibody that binds IL-23/p19, thereby preparing a hybridhybridoma that secretes a bispecific monoclonal antibody that crossbinds with IL-17A or IL-17F and also binds IL-23p19 monoclonal antibody.In one embodiment, the method comprises fusing hybridoma cells thatsecrete an antagonistic (or agonistic) IL-17A or IL-17F MAb, withhybridoma cells that secrete an antagonistic (or agonistic) IL-23/p19MAb. Conventional techniques for conducting such a fusion, and forisolating the desired hybrid hybridoma, include those describedelsewhere herein, and those illustrated in the examples below.

U.S. Pat. No. 6,060,285 discloses a process for the production ofbispecific antibodies, in which at least the genes for the light chainand the variable portion of the heavy chain of an antibody having afirst specificity are transfected into a hybridoma cell secreting anantibody having a second specificity. When the transfected hybridomacells are cultured, bispecific antibodies are produced, and may beisolated by various means known in the art.

Other investigators have used chemical coupling of antibody fragments toprepare antigen-binding molecules having specificity for two differentantigens (Brennan et al., Science 229:81 1985; Glennie et al., J.Immunol. 139:2367, 1987). U.S. Pat. No. 6,010,902 also discussestechniques known in the art by which bispecific antibodies can beprepared, for example by the use of heterobifunctional cross-linkingreagents such as GMBS (maleimidobutyryloxy succinimide) or SPDP(N-succinimidyl 3-(2-pyridyldithio)propionate). (See, e.g., Hardy,“Purification And Coupling Of Fluorescent Proteins For Use In FlowCytometry”, Handbook Of Experimental Immunology, 4.sup.th Ed., Volume 1,Immunochemistry, Weir et al. (eds.), pp. 31.4-31.12, 1986).

The ability to produce antibodies via recombinant DNA technology hasfacilitated production of bispecific antibodies. Kostelny et al.utilized the leucine zipper moieties from the fos and jun proteins(which preferentially form heterodimers) to produce bispecificantibodies able to bind both the cell surface molecule CD3 and thereceptor for IL-2 (J. Immunol. 148:1547; 1992).

Single chain antibodies may be formed by linking heavy and light chainvariable region (Fv region) fragments via an amino acid bridge (shortpeptide linker), resulting in a single polypeptide chain. Suchsingle-chain Fvs (scFvs) have been prepared by fusing DNA encoding apeptide linker between DNAs encoding the two variable regionpolypeptides (V_(L) and V_(H)). The resulting antibody fragments canform dimers or higher oligomers, depending on such factors as the lengthof a flexible linker between the two variable domains (Kortt et al.,Protein Engineering 10:423, 1997). In particular embodiments, two ormore scFvs are joined by use of a chemical cross-linking agent.

Techniques developed for the production of single chain antibodies canbe adapted to produce single chain antibodies of the present inventionthat bind both IL-17A or IL-17F and IL-23. Such techniques include thosedescribed in U.S. Pat. No. 4,946,778; Bird (Science 242:423, 1988);Huston et al. (Proc. Natl. Acad. Sci. USA 85:5879, 1988); and Ward etal. (Nature 334:544, 1989). Once desired single chain antibodies areidentified (for example, from a phage-display library), those of skillin the art can further manipulate the DNA encoding the single chainantibody(ies) to yield bispecific antibodies, including bispecificantibodies having Fc regions.

Single chain antibodies against IL-17A or IL-17F and IL-23 may beconcatamerized in either order (i.e., anti-IL-17A-anti-IL-23 oranti-IL-23-anti-IL-17A). In particular embodiments, starting materialsfor preparing a bispecific antibody include an antagonistic (oragonistic) single chain antibody directed against IL-17A or againstIL-17F and an antagonistic (or agonistic) single chain antibody directedagainst IL-23/p19.

The scFv entities that bind IL-17A or IL-17F and IL-23p19 can beoriented with the variable light region either amino terminal to thevariable heavy region or carboxyl terminal to it. Additionally, tandemscFvs can be prepared in a number of configurations, such that eachtarget, i.e, IL-17A or IL-17F and IL-23p19 can be bound by itsrespective variable regions. Thus, the construct for a tandem scFVmolecule can be prepared such that the variable light region andvariable heavy region of one antibody can be interspersed with thevariable light and variable heavy regions of the other antibody as longas the variable regions are able to bind the targets. Tandem scFvmolecules that bind both targets can be prepared with a linker betweenthe scFv entities, including a Gly-Ser linker comprising a series ofglycine and serine residues and can also include additional amino acids.

U.S. Pat. No. 5,582,996 discloses the use of complementary interactivedomains (such as leucine zipper moieties or other lock and keyinteractive domain structures) to facilitate heterodimer formation inthe production of bispecific antibodies. The complementary interactivedomain(s) may be inserted between an Fab fragment and another portion ofa heavy chain (i.e., C.sub.H1 or C.sub.H2 regions of the heavy chain).The use of two different Fab fragments and complementary interactivedomains that preferentially heterodimerize will result in bispecificantibody molecules. Cysteine residues may be introduced into thecomplementary interactive domains to allow disulphide bonding betweenthe complementary interactive domains and stabilize the resultingbispecific antibodies.

Tetravalent, bispecific molecules can be prepared by fusion of DNAencoding the heavy chain of an F(ab′)₂ fragment of an antibody witheither DNA encoding the heavy chain of a second F(ab′)₂ molecule (inwhich the CH1 domain is replaced by a CH3 domain), or with DNA encodinga single chain Fv fragment of an antibody, as described in U.S. Pat. No.5,959,083. Expression of the resultant fusion genes in mammalian cells,together with the genes for the corresponding light chains, yieldstetravalent bispecific molecules having specificity for selectedantigens.

Bispecific antibodies can also be produced as described in U.S. Pat. No.5,807,706, which is incorporated by reference herein. Generally, themethod involves introducing a protuberance in a first polypeptide and acorresponding cavity in a second polypeptide, polypeptides interface.The protuberance and cavity are positioned so as to promoteheteromultimer formation and hinder homomultimer formation. Theprotuberance is created by replacing amino acids having small sidechains with amino acids having larger side chains. The cavity is createdby the opposite approach, i.e., replacing amino acids having relativelylarge side chains with amino acids having smaller side chains.

The protuberance and cavity can be generated by conventional methods formaking amino acid substitutions in polypeptides. For example, a nucleicacid encoding a polypeptide may be altered by conventional in vitromutagenesis techniques. Alternatively, a polypeptide incorporating adesired amino acid substitution may be prepared by peptide synthesis.Amino acids chosen for substitution are located at the interface betweenthe first and second polypeptides.

Screening for antibodies that specifically bind to IL-17A or IL-17F andIL-23/p19 may be accomplished using an enzyme-linked immunosorbant assay(ELISA) in which microtiter plates are coated with IL-17A or IL-17F andIL-23 (or p19 alone). In some embodiments, antibodies that bind bothIL-17A or IL-17F and IL-23/p19 from positively reacting clones can befurther screened for reactivity in an ELISA-based assay using microtiterplates coated with the other forms IL-17 and IL-23/p19, or other IL-17family members. Clones that produce antibodies that are reactive toanother forms or family members are eliminated, and clones that produceantibodies that are reactive to both IL-17A or IL-17F and IL-23/p19 maybe selected for further expansion. Confirmation of reactivity of theantibodies to both IL-17A or IL-17F and IL-23/p19 may be accomplished,for example, using a Western Blot assay in which protein from ovarian,breast, renal, colorectal, lung, endometrial, or brain cancer cells andpurified FR-.alpha. and other folate receptor isoforms are run on anSDS-PAGE gel, and subsequently are blotted onto a membrane. The membranemay then be probed with the putative anti-FR-.alpha. antibodies.Reactivity with both IL-17A or IL-17F and IL-23/p19 and not anotherfamily member confirms specificity of reactivity for IL-17A/Fcross-binding antibodies and IL-23/p19.

Antibody-producing cells of the invention include any insect expressioncell line known, such as for example, Spodoptera frugiperda cells. Theexpression cell lines may also be yeast cell lines, such as, forexample, Saccharomyces cerevisiae and Schizosaccharomyces pombe cells.The expression cells may also be mammalian cells such as, for example,hybridoma cells (e.g., NS0 cells), Chinese hamster ovary cells, babyhamster kidney cells, human embryonic kidney line 293, normal dog kidneycell lines, normal cat kidney cell lines, monkey kidney cells, Africangreen monkey kidney cells, COS cells, and non-tumorigenic mouse myoblastG8 cells, fibroblast cell lines, myeloma cell lines, mouse NIH/3T3cells, LMTK31 cells, mouse sertoli cells, human cervical carcinomacells, buffalo rat liver cells, human lung cells, human liver cells,mouse mammary tumor cells, TRI cells, MRC 5 cells, and FS4 cells.

In some preferred embodiments, the antibody-producing cells of theinvention produce antibodies that specifically bind to IL-17A or IL-17Fand IL-23/p19 (either singly or together as with a bispecific antibodyor scFV). The cells preferably are substantially free of IL-17A, IL-17Fand IL-23 binding competitors. In preferred embodiments, theantibody-producing cells comprise less than about 10%, preferably lessthan about 5%, more preferably less than about 1%, more preferably lessthan about 0.5%, more preferably less than about 0.1%, and mostpreferably 0% by weight IL-17A, IL-17F, or IL-23 binding competitors. Insome embodiments, the antibodies produced by the antibody-producingcells are substantially free of IL-17A, IL-17F, and IL-23 competitors.In preferred embodiments, antibodies produced by the antibody-producingcells comprise less than about 10%, preferably less than about 5%, morepreferably less than about 1%, more preferably less than about 0.5%,more preferably less than about 0.1%, and most preferably 0% by weightboth IL-17 and IL-23 binding competitors. P

Methods of antibody purification are known in the art. In someembodiments of the invention, methods for antibody purification includefiltration, affinity column chromatography, cation exchangechromatography, anion exchange chromatography, and concentration. Thefiltration step preferably comprises ultrafiltration, and morepreferably ultrafiltration and diafiltration. Filtration is preferablyperformed at least about 5-50 times, more preferably 10 to 30 times, andmost preferably 14 to 27 times. Affinity column chromatography, may beperformed using, for example, PROSEP Affinity Chromatography (Millipore,Billerica, Mass.). In a preferred embodiment, the affinitychromatography step comprises PROSEP-VA column chromatography. Eluatemay be washed in a solvent detergent. Cation exchange chromatography mayinclude, for example, SP-Sepharose Cation Exchange Chromatography. Anionexchange chromatography may include, for example but not limited to,Q-Sepharose Fast Flow Anion Exchange. The anion exchange step ispreferably non-binding, thereby allowing removal of contaminantsincluding DNA and BSA. The antibody product is preferably nanofiltered,for example, using a Pall DV 20 Nanofilter. The antibody product may beconcentrated, for example, using ultrafiltration and diafiltration. Themethod may further comprise a step of size exclusion chromatography toremove aggregates.

Antibodies that bind to both IL-17A or IL-17F and IL-23 can be used tomodulate the immune system by binding IL-17A or IL-17F and IL-23/p19(either singly or together as with a bispecific antibody or scFV), andthus, preventing the binding of IL-17A or IL-17F with either IL-17RA orIL-17RC and IL-23 with its receptor or any other receptor that they maybind. The antibodies of the invention can also be used to modulate theimmune system by inhibiting the binding of both IL-17A or IL-17F withthe endogenous IL-17RA and/or IL-17RC receptor and IL-23 with itsendogenous receptor. The antibodies of the invention can be also used totreat a subject which produces an excess of either IL-17A or IL-17Fand/or IL-23. Suitable subjects include mammals, such as humans. Forexample, the antibodies of the invention are useful in binding,blocking, inhibiting, reducing, antagonizing or neutralizing of bothIL-17A or IL-17F and IL-23 (either singly or together as with abispecific antibody or scFV), in the treatment of inflammation andinflammatory diseases such as multiple sclerosis, cancer (characterizedby IL-17A or IL-17F and IL-23 expression), psoriasis, psoriaticarthritis, atopic dermatitis, inflammatory skin conditions, rheumatoidarthritis, inflammatory bowel disease (IBD), Crohn's Disease,diverticulosis, asthma, pancreatitis, type I diabetes (IDDM), pancreaticcancer, pancreatitis, Graves Disease, colon and intestinal cancer,autoimmune disease, sepsis, organ or bone marrow transplant;inflammation due to endotoxemia, trauma, surgery or infection;amyloidosis; splenomegaly; graft versus host disease; and whereinhibition of inflammation, immune suppression, reduction ofproliferation of hematopoietic, immune, inflammatory or lymphoid cells,macrophages, T-cells (including Th1 and Th2 cells), suppression ofimmune response to a pathogen or antigen, or other instances whereinhibition of IL-17F and IL-17A cytokines is desired.

Within preferred embodiments, the antibodies of the invention bind to,blocks, inhibits, reduces, antagonizes or neutralizes IL-23 (via p19)and IL-17A IL-17F either singly or together as with a bispecificantibody or scFV), in vivo.

Moreover, the antibodies of the invention are useful to:

(1) Block, inhibit, reduce, antagonize or neutralize signaling viaIL-17A or IL-17F and IL-23 in the treatment of cancer, acuteinflammation, and chronic inflammatory diseases such as inflammatorybowel disease (IBD), IBS, chronic colitis, splenomegaly, rheumatoidarthritis, and other diseases associated with the induction ofacute-phase response.

(2) Block, inhibit, reduce, antagonize or neutralize signaling via IL-17A or IL-17F or IL-23 in the treatment of autoimmune diseases such asinsulin-dependent diabetes mellitus (IDDM), multiple sclerosis (MS),systemic Lupus erythematosus (SLE), myasthenia gravis, rheumatoidarthritis, IBS and IBD to prevent or inhibit signaling in immune cells(e.g. lymphocytes, monocytes, leukocytes) via their receptors (e.g.IL-17RA and IL-17RC). Blocking, inhibiting, reducing, or antagonizingsignaling via IL-17RA and IL-17RC, using the antibodies of the presentinvention, may also benefit diseases of the pancreas, kidney, pituitaryand neuronal cells. IDDM, non-insulin dependent diabetes mellitus(NIDDM), pancreatitis, and pancreatic carcinoma may benefit.

(3) Agonize, enhance, increase or initiate signaling via IL-17A orIL-17F receptors in the treatment of autoimmune diseases such as IDDM,MS, SLE, myasthenia gravis, rheumatoid arthritis, IBS, and IBD.Anti-IL-17A and IL-17F neutralizing and monoclonal antibodies may signallymphocytes or other immune cells to differentiate, alter proliferation,or change production of cytokines or cell surface proteins thatameliorate autoimmunity. Specifically, modulation of a T-helper cellresponse to an alternate pattern of cytokine secretion may deviate anautoimmune response to ameliorate disease (Smith J A et al., J. Immunol.160:4841-4849, 1998). Similarly, agonistic antibodies may be used tosignal, deplete and deviate immune cells involved in asthma, allergy andatopic disease. Signaling via IL-17RA and IL-17RC may also benefitdiseases of the pancreas, kidney, pituitary and neuronal cells. IDDM,NIDDM, pancreatitis, and pancreatic carcinoma may benefit.

The antibodies described herein can be used to bind, block, inhibit,reduce, antagonize or neutralize IL-23 and IL-17 A or IL-17F activity,either singly or together as with a bispecific antibody or scFV, in thetreatment of multiple sclerosis, cancer, autoimmune disease, atopicdisease, NIDDM, pancreatitis and kidney dysfunction as described above.The antibodies of the present invention are useful as antagonists ofIL-17 A or IL-17F or IL-23. Such antagonistic effects can be achieved bydirect neutralization or binding of IL-17 A or IL-17F and IL-23 (viap19).

Antibodies herein can also be directly or indirectly conjugated todrugs, toxins, radionuclides and the like, and these conjugates used forin vivo diagnostic or therapeutic applications. For instance, antibodiesor binding polypeptides which recognize IL-17A or IL-17F or IL-23 can beused to identify or treat tissues or organs that express a correspondinganti-complementary molecule. More specifically, antibodies to IL-17 orIL-23 or bioactive fragments or portions thereof, can be coupled todetectable or cytotoxic molecules and delivered to a mammal havingcells, tissues or organs that express these cytokines IL-17 orIL-23-expressing cancers.

Suitable detectable molecules may be directly or indirectly attached tothe antagonists of the present invention, such as “bindingpolypeptides,” (including binding peptides disclosed above), antibodies,or bioactive fragments or portions thereof. Suitable detectablemolecules include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent markers, chemiluminescent markers, magneticparticles and the like. Suitable cytotoxic molecules may be directly orindirectly attached to the polypeptide or antibody, and includebacterial or plant toxins (for instance, diphtheria toxin, Pseudomonasexotoxin, ricin, abrin and the like), as well as therapeuticradionuclides, such as iodine-131, rhenium-188 or yttrium-90 (eitherdirectly attached to the polypeptide or antibody, or indirectly attachedthrough means of a chelating moiety, for instance). Binding polypeptidesor antibodies may also be conjugated to cytotoxic drugs, such asadriamycin. For indirect attachment of a detectable or cytotoxicmolecule, the detectable or cytotoxic molecule can be conjugated with amember of a complementary/anticomplementary pair, where the other memberis bound to the binding polypeptide or antibody portion. For thesepurposes, biotin/streptavidin is an exemplarycomplementary/anticomplementary pair.

In another embodiment, binding polypeptide-toxin fusion proteins orantibody-toxin fusion proteins can be used for targeted cell or tissueinhibition or ablation (for instance, to treat cancer cells or tissues).Alternatively, if the binding polypeptide has multiple functionaldomains (i.e., an activation domain or a ligand binding domain, plus atargeting domain), a fusion protein including only the targeting domainmay be suitable for directing a detectable molecule, a cytotoxicmolecule or a complementary molecule to a cell or tissue type ofinterest. In instances where the fusion protein including only a singledomain includes a complementary molecule, the anti-complementarymolecule can be conjugated to a detectable or cytotoxic molecule. Suchdomain-complementary molecule fusion proteins thus represent a generictargeting vehicle for cell/tissue-specific delivery of genericanti-complementary-detectable/cytotoxic molecule conjugates.

Inflammation is a protective response by an organism to fend off aninvading agent. Inflammation is a cascading event that involves manycellular and humoral mediators. On one hand, suppression of inflammatoryresponses can leave a host immunocompromised; however, if leftunchecked, inflammation can lead to serious complications includingchronic inflammatory diseases (e.g., psoriasis, arthritis, rheumatoidarthritis, multiple sclerosis, inflammatory bowel disease and the like),septic shock and multiple organ failure. Importantly, these diversedisease states share common inflammatory mediators. The collectivediseases that are characterized by inflammation have a large impact onhuman morbidity and mortality. Therefore it is clear thatanti-inflammatory proteins, such as antagonists to IL-17A or IL-17F andIL-23/p19, such as IL-17A or IL-17F and IL-23/p19 antibodies, could havecrucial therapeutic potential for a vast number of human and animaldiseases, from asthma and allergy to autoimmunity, cancers, and septicshock.

Arthritis

Arthritis, including osteoarthritis, rheumatoid arthritis, arthriticjoints as a result of injury, and the like, are common inflammatoryconditions which would benefit from the therapeutic use ofanti-inflammatory proteins, such as the antagonists of the presentinvention. For example, rheumatoid arthritis (RA) is a systemic diseasethat affects the entire body and is one of the most common forms ofarthritis. It is characterized by the inflammation of the membranelining the joint, which causes pain, stiffness, warmth, redness andswelling. Inflammatory cells release enzymes that may digest bone andcartilage. As a result of rheumatoid arthritis, the inflamed jointlining, the synovium, can invade and damage bone and cartilage leadingto joint deterioration and severe pain amongst other physiologiceffects. The involved joint can lose its shape and alignment, resultingin pain and loss of movement.

Rheumatoid arthritis (RA) is an immune-mediated disease particularlycharacterized by inflammation and subsequent tissue damage leading tosevere disability and increased mortality. A variety of cytokines areproduced locally in the rheumatoid joints. Numerous studies havedemonstrated that IL-1 and TNF-alpha, two prototypic pro-inflammatorycytokines, play an important role in the mechanisms involved in synovialinflammation and in progressive joint destruction. Indeed, theadministration of TNF-alpha and IL-1 inhibitors in patients with RA hasled to a dramatic improvement of clinical and biological signs ofinflammation and a reduction of radiological signs of bone erosion andcartilage destruction. However, despite these encouraging results, asignificant percentage of patients do not respond to these agents,suggesting that other mediators are also involved in the pathophysiologyof arthritis (Gabay, Expert. Opin. Biol. Ther. 2(2):135-149 (2002). Oneof those mediators could be IL-17 or IL-23, as demonstrated in severalreports to play a role in rheumatoid arthritis. For example, IL-17 andIL-23/p19 are overexpressed in the synovium and synovial fibroblasts ofpatients with rheumatoid arthritis compared to individuals withoutrheumatoid arthritis. Furthermore, IL-17 and IL-23/p19 have beendemonstrated to promote matrix degradation and enhance the expression ofinflammatory, matrix-destructive cytokines when added tosynovium/synoviocyte cultures. (Murphy et al, J. Exp. Med 198:1951(2003); reviewed in Lubberts et al, Arthritis Res Ther. 7:29 (2005) andKim et al, Rheumatology, Jun. 12, 2006 (online publication ahead ofprint)). Therefore, such a molecule that binds or inhibits IL-17 orIL-23 activity, such as the antagonists of the present invention, couldserve as a valuable therapeutic to reduce inflammation in rheumatoidarthritis, and other arthritic diseases.

There are several animal models for rheumatoid arthritis known in theart. For example, in the collagen-induced arthritis (CIA) model, micedevelop chronic inflammatory arthritis that closely resembles humanrheumatoid arthritis. Since CIA shares similar immunological andpathological features with RA, this makes it an ideal model forscreening potential human anti-inflammatory compounds. The CIA model isa well-known model in mice that depends on both an immune response, andan inflammatory response, in order to occur. The immune responsecomprises the interaction of B-cells and CD4+ T-cells in response tocollagen, which is given as antigen, and leads to the production ofanti-collagen antibodies. The inflammatory phase is the result of tissueresponses from mediators of inflammation, as a consequence of some ofthese antibodies cross-reacting to the mouse's native collagen andactivating the complement cascade. An advantage in using the CIA modelis that the basic mechanisms of pathogenesis are known. The relevantT-cell and B-cell epitopes on type II collagen have been identified, andvarious immunological (e.g., delayed-type hypersensitivity andanti-collagen antibody) and inflammatory (e.g., cytokines, chemokines,and matrix-degrading enzymes) parameters relating to immune-mediatedarthritis have been determined, and can thus be used to assess testcompound efficacy in the CIA model (Wooley, Curr. Opin. Rheum. 3:407-20(1999); Williams et al., Immunol. 89:9784-788 (1992); Myers et al., LifeSci. 61:1861-78 (1997); and Wang et al., Immunol. 92:8955-959 (1995)).

One group has shown that an anti-mouse IL-17 antibody reduces symptomsin a mouse CIA-model relative to control mice, and another group hasshown that deficiency of IL-23/p19 is protective in CIA (Murphy et al,J. Exp. Med 198:1951 (2003)), thus showing conceptually that antagonistsof the present invention may be beneficial in treating human disease.The administration of a single mouse-IL-17-specific rat antisera reducedthe symptoms of arthritis in the animals when introducedprophylactically or after symptoms of arthritis were already present inthe model (Lubberts et al, Arthritis Rheum. 50:650-9 (2004)).

As described in the Examples below, both IL-17 and IL-23/p19 areoverexpressed in CIA. Therefore, antagonists of the present inventioncan be used to neutralize IL-17 and/or IL-23 (via p19) in the treatmentof specific human diseases such as arthritis, psoriasis, psoriaticarthritis, endotoxemia, inflammatory bowel disease (IBD), IBS, colitis,and other inflammatory conditions disclosed herein.

The administration of antagonists of the present invention to these CIAmodel mice is used to evaluate the use of these antagonists toameliorate symptoms and alter the course of disease. Moreover, resultsshowing inhibition of IL-17 and/or IL-23 signalling by these antagonistswould provide proof of concept that IL-17 and IL-23/p19 antagonists,such as those disclosed herein, can also be used to ameliorate symptomsand alter the course of disease. By way of example and withoutlimitation, the injection of 10-200 ug of an anti-IL-17 andanti-IL-23/p19 per mouse (one to seven times a week for up to but notlimited to 4 weeks via s.c., i.p., or i.m route of administration) cansignificantly reduce the disease score (paw score, incident ofinflammation, or disease). Depending on the initiation of administration(e.g. prior to or at the time of collagen immunization, or at any timepoint following the second collagen immunization, including those timepoints at which the disease has already progressed), antagonists of thepresent invention can be efficacious in preventing rheumatoid arthritis,as well as preventing its progression.

Endotoxemia

Endotoxemia is a severe condition commonly resulting from infectiousagents such as bacteria and other infectious disease agents, sepsis,toxic shock syndrome, or in immunocompromised patients subjected toopportunistic infections, and the like. Therapeutically useful ofanti-inflammatory proteins, such as antibodies of the invention, couldaid in preventing and treating endotoxemia in humans and animals. Suchantibodies could serve as a valuable therapeutic to reduce inflammationand pathological effects in endotoxemia.

Inflammatory Bowel Disease IBD

In the United States approximately 500,000 people suffer fromInflammatory Bowel Disease (IBD) which can affect either colon andrectum (Ulcerative colitis) or both, small and large intestine (Crohn'sDisease). In both Crohn's disease and ulcerative colitis, the tissuedamage results from an inappropriate or exaggerated immune response toantigens of the gut microflora. This review summarizes current knowledgeregarding the role of immune-inflammatory mediators in the pathogenesisof inflammatory bowel disease. Despite having a common basis in overresponsiveness to luminal antigens, Crohn's disease and ulcerativecolitis are immunologically distinct entities. Crohn's disease isassociated with a Th1 T cell-mediated response, characterized byenhanced production of interferon-[gamma] and tumor necrosisfactor-[alpha]. Interleukin (IL)-12 and, possibly, IL-23 govern the Th1cell differentiation, but optimal induction and stabilization ofpolarized Th1 cells would require additional cytokines, such as IL-15,IL-18 and IL-21. In ulcerative colitis, the local immune response isless polarized, but it is characterized by CD1-reactive natural killer Tcell production of IL-13. Beyond these differences, Crohn's disease andulcerative colitis share important end-stage effector pathways ofintestinal injury, which are mediated by an active cross-talk betweenimmune and non-immune mucosal cells. As shown in the Examples below,IL-17 and IL-23 are both overexpressed in intestines and/or serum fromhumans with IBD and in mouse models of IBD. Moreover, neutralization ofIL-17 and/or IL-23/p19 can reduced disease symptoms and pathology inanimals models of IBD (Nielson et al, Scand J Gastroenterol. 38:180(2003); Schmidt et al, Inflamm. Bowel Dis. 11:16 (2005); Fuss et al,Inflamm Bowel Dis. 12:9 (2006)). Moreover, neutralization of IL-17and/or IL-23/p19 can reduce disease symptoms and pathology in animalsmodels of IBD (Yen et al, J. Clin. Invest. 116:1310 (2006); Zhang et al,Inflamm Bowel Dis. 12:382 (2006)).

As shown in the Examples below, both IL-17 and IL-23/p19 expression isincreased in DSS colitis. Thus, antagonists of the present inventioncould serve as a valuable therapeutic to reduce inflammation andpathological effects in IBD and related diseases.

Ulcerative colitis (UC) is an inflammatory disease of the largeintestine, commonly called the colon, characterized by inflammation andulceration of the mucosa or innermost lining of the colon. Thisinflammation causes the colon to empty frequently, resulting indiarrhea. Symptoms include loosening of the stool and associatedabdominal cramping, fever and weight loss.

Although the exact cause of UC is unknown, recent research suggests thatthe body's natural defenses are operating against proteins in the bodywhich the body thinks are foreign (an “autoimmune reaction”). Perhapsbecause they resemble bacterial proteins in the gut, these proteins mayeither instigate or stimulate the inflammatory process that begins todestroy the lining of the colon. As the lining of the colon isdestroyed, ulcers form releasing mucus, pus and blood. The diseaseusually begins in the rectal area and may eventually extend through theentire large bowel. Repeated episodes of inflammation lead to thickeningof the wall of the intestine and rectum with scar tissue. Death of colontissue or sepsis may occur with severe disease. The symptoms ofulcerative colitis vary in severity and their onset may be gradual orsudden. Attacks may be provoked by many factors, including respiratoryinfections or stress.

Although there is currently no cure for UC available, treatments arefocused on suppressing the abnormal inflammatory process in the colonlining. Treatments including corticosteroids immunosuppressives (eg.azathioprine, mercaptopurine, and methotrexate) and aminosalicytates areavailable to treat the disease. However, the long-term use ofimmunosuppressives such as corticosteroids and azathioprine can resultin serious side effects including thinning of bones, cataracts,infection, and liver and bone marrow effects. In the patients in whomcurrent therapies are not successful, surgery is an option. The surgeryinvolves the removal of the entire colon and the rectum.

There are several animal models that can partially mimic chroniculcerative colitis. One of the most widely used models is the2,4,6-trinitrobenesulfonic acid/ethanol (TNBS) induced colitis model,which induces chronic inflammation and ulceration in the colon. WhenTNBS is introduced into the colon of susceptible mice via intra-rectalinstillation, it induces T-cell mediated immune response in the colonicmucosa, in this case leading to a massive mucosal inflammationcharacterized by the dense infiltration of T-cells and macrophagesthroughout the entire wall of the large bowel. Moreover, thishistopathologic picture is accompanies by the clinical picture ofprogressive weight loss (wasting), bloody diarrhea, rectal prolapse, andlarge bowel wall thickening (Neurath et al. Intern. Rev. Immunol.19:51-62, 2000).

Another colitis model uses dextran sulfate sodium (DSS), which inducesan acute colitis manifested by bloody diarrhea, weight loss, shorteningof the colon and mucosal ulceration with neutrophil infiltration.DSS-induced colitis is characterized histologically by infiltration ofinflammatory cells into the lamina propria, with lymphoid hyperplasia,focal crypt damage, and epithelial ulceration. These changes are thoughtto develop due to a toxic effect of DSS on the epithelium and byphagocytosis of lamina propria cells and production of TNF-alpha andIFN-gamma. Despite its common use, several issues regarding themechanisms of DSS about the relevance to the human disease remainunresolved. DSS is regarded as a T cell-independent model because it isobserved in T cell-deficient animals such as SCID mice.

The administration of antagonists of the present invention to these TNBSor DSS models can be used to evaluate the use of those antagonists toameliorate symptoms and alter the course of gastrointestinal disease.Moreover, the results showing inhibition of IL-17 or IL-23 signallingprovide proof of concept that other IL-17/IL-23 antagonists can also beused to ameliorate symptoms in the colitis/IBD models and alter thecourse of disease.

Psoriasis

Psoriasis is a chronic skin condition that affects more than sevenmillion Americans. Psoriasis occurs when new skin cells grow abnormally,resulting in inflamed, swollen, and scaly patches of skin where the oldskin has not shed quickly enough. Plaque psoriasis, the most commonform, is characterized by inflamed patches of skin (“lesions”) toppedwith silvery white scales. Psoriasis may be limited to a few plaques orinvolve moderate to extensive areas of skin, appearing most commonly onthe scalp, knees, elbows and trunk. Although it is highly visible,psoriasis is not a contagious disease. The pathogenesis of the diseasesinvolves chronic inflammation of the affected tissues. IL-17 and IL-23are both overexpressed in psoriatic skin compared to non-psoriatic skin(Li et al, J Huazhong Univ Sci Technolog Med Sci. 24:294 (2004); Piskinet al, J Immunol. 176:1908 (2006)). Therefore, antagonists of thepresent invention could serve as a valuable therapeutic to reduceinflammation and pathological effects in psoriasis, other inflammatoryskin diseases, skin and mucosal allergies, and related diseases.

Psoriasis is a T-cell mediated inflammatory disorder of the skin thatcan cause considerable discomfort. It is a disease for which there is nocure and affects people of all ages. Psoriasis affects approximately twopercent of the populations of European and North America. Althoughindividuals with mild psoriasis can often control their disease withtopical agents, more than one million patients worldwide requireultraviolet or systemic immunosuppressive therapy. Unfortunately, theinconvenience and risks of ultraviolet radiation and the toxicities ofmany therapies limit their long-term use. Moreover, patients usuallyhave recurrence of psoriasis, and in some cases rebound, shortly afterstopping immunosuppressive therapy.

Antibodies that bind IL-17A and IL-17F may also be used withindiagnostic systems for the detection of circulating levels of IL-17F orIL-17A, and in the detection of IL-17A and/or IL-17F associated withacute phase inflammatory response. Elevated or depressed levels ofligand or receptor polypeptides may be indicative of pathologicalconditions, including inflammation or cancer. IL-17A and IL-17F areknown to induce associated acute phase inflammatory response. Moreover,detection of acute phase proteins or molecules such as IL-17A or IL-17Fcan be indicative of a chronic inflammatory condition in certain diseasestates (e.g., asthma, psoriasis, rheumatoid arthritis, colitis, IBD).Detection of such conditions serves to aid in disease diagnosis as wellas help a physician in choosing proper therapy.

In addition to other disease models described herein, the activity ofantagonists of the present invention on inflammatory tissue derived fromhuman psoriatic lesions can be measured in vivo using a severe combinedimmune deficient (SCID) mouse model. Several mouse models have beendeveloped in which human cells are implanted into immunodeficient mice(collectively referred to as xenograft models); see, for example, CattanA R, Douglas E, Leuk. Res. 18:513-22 (1994) and Flavell, D J,Hematological Oncology 14:67-82 (1996). As an in vivo xenograft modelfor psoriasis, human psoriatic skin tissue is implanted into the SCIDmouse model, and challenged with an appropriate antagonist. Moreover,other psoriasis animal models in the art may be used to evaluate thepresent antagonists, such as human psoriatic skin grafts implanted intoAGR129 mouse model, and challenged with an appropriate antagonist (e.g.,see, Boyman, O. et al., J. Exp. Med. Online publication #20031482, 2004,incorporated herein by reference). IL-17/IL-23 antibodies or bindingpeptides that bind, block, inhibit, reduce, antagonize or neutralize theactivity of IL-17, IL-23 or both IL-17 and IL-23 are preferredantagonists. Similarly, tissues or cells derived from human colitis,IBD, arthritis, or other inflammatory lesions can be used in the SCIDmodel to assess the anti-inflammatory properties of the IL-17 and IL-23antagonists described herein.

Therapies designed to abolish, retard, or reduce inflammation usingantibodies of the invention can be tested by administration of suchantibodies to SCID mice bearing human inflammatory tissue (e.g.,psoriatic lesions and the like), or other models described herein.Efficacy of treatment is measured and statistically evaluated asincreased anti-inflammatory effect within the treated population overtime using methods well known in the art. Some exemplary methodsinclude, but are not limited to measuring for example, in a psoriasismodel, epidermal thickness, the number of inflammatory cells in theupper dermis, and the grades of parakeratosis. Such methods are known inthe art and described herein. For example, see Zeigler, M. et al. LabInvest 81:1253 (2001); Zollner, T. M. et al. J. Clin. Invest. 109:671(2002); Yamanaka, N. et al. Microbiol. Immunol. 45:507 (2001);Raychaudhuri, S. P. et al. Br. J. Dermatol. 144:931 (2001); Boehncke, W.H et al. Arch. Dermatol. Res. 291:104, (1999); Boehncke, W. H et al. J.Invest. Dermatol. 116:596 (2001); Nickoloff, B. J. et al. Am. J. Pathol.146:580 (1995); Boehncke, W. H et al. J. Cutan. Pathol. 24:1, (1997);Sugai, J., M. et al. J. Dermatol. Sci. 17:85 (1998); and Villadsen L. S.et al. J. Clin. Invest. 112:1571 (2003). Inflammation may also bemonitored over time using well-known methods such as flow cytometry (orPCR) to quantitate the number of inflammatory or lesional cells presentin a sample, score (weight loss, diarrhea, rectal bleeding, colonlength) for IBD. For example, therapeutic strategies appropriate fortesting in such a model include direct treatment using IL-17 and IL-23antagonists (singly or together), or related conjugates or antagonistsbased on the disrupting interaction of IL-17 and IL-23 with theirreceptors.

Moreover, Psoriasis is a chronic inflammatory skin disease that isassociated with hyperplastic epidermal keratinocytes and infiltratingmononuclear cells, including CD4+ memory T cells, neutrophils andmacrophages (Christophers, Int. Arch. Allergy Immunol., 110:199, 1996).It is currently believed that environmental antigens play a significantrole in initiating and contributing to the pathology of the disease.However, it is the loss of tolerance to self-antigens that is thought tomediate the pathology of psoriasis. Dendritic cells and CD4+ T cells arethought to play an important role in antigen presentation andrecognition that mediate the immune response leading to the pathology.We have recently developed a model of psoriasis based on the CD4+CD45RBtransfer model (Davenport et al., Internat. Immunopharmacol.,2:653-672). Antibodies of the present invention are administered to themice. Inhibition of disease scores (skin lesions, inflammatorycytokines) indicates the effectiveness of such antibodies in psoriasis.

Atopic Dermatitis.

AD is a common chronic inflammatory disease that is characterized byhyperactivated cytokines of the helper T cell subset 2 (Th2). Althoughthe exact etiology of AD is unknown, multiple factors have beenimplicated, including hyperactive Th2 immune responses, autoimmunity,infection, allergens, and genetic predisposition. Key features of thedisease include xerosis (dryness of the skin), pruritus (itchiness ofthe skin), conjunctivitis, inflammatory skin lesions, Staphylococcusaureus infection, elevated blood eosinophilia, elevation of serum IgEand IgG1, and chronic dermatitis with T cell, mast cell, macrophage andeosinophil infiltration. Colonization or infection with S. aureus hasbeen recognized to exacerbate AD and perpetuate chronicity of this skindisease.

AD is often found in patients with asthma and allergic rhinitis, and isfrequently the initial manifestation of allergic disease. About 20% ofthe population in Western countries suffer from these allergic diseases,and the incidence of AD in developed countries is rising for unknownreasons. AD typically begins in childhood and can often persist throughadolescence into adulthood. Current treatments for AD include topicalcorticosteroids, oral cyclosporin A, non-corticosteroidimmunosuppressants such as tacrolimus (FK506 in ointment form), andinterferon-gamma. Despite the variety of treatments for AD, manypatients' symptoms do not improve, or they have adverse reactions tomedications, requiring the search for other, more effective therapeuticagents. The antagonists of the present invention can be used toneutralize IL-17 and IL-23 (via p19) in the treatment of specific humandiseases such as atopic dermatitis, inflammatory skin conditions, andother inflammatory conditions disclosed herein.

Asthma

IL-17 plays an important role in allergen-induced T cell activation andneutrophilic influx in the airways. The receptor for IL-17 is expressedin the airways (Yao, et al. Immunity 3:811 (1995)) and IL-17 mediatedneutrophil recruitment in allergic asthma is largely induced by thechemoattractant IL-8, GRO-Q and macrophage inflammatory protein-2(MIP-2) produced by IL-17 stimulated human bronchial epithelial cells(HBECs) and human bronchial fibroblasts (Yao, et al. J Immunol 155:5483(1995)); Molet, et al. J Allergy Clin Immunol 108:430 (2001)). IL-17also stimulates HBECs to release IL-6, a neutrophil-activating factor(Fossiez, et al, J Exp Med 183:2593 (1996), and Linden, et al. Int ArchAllergy Immunol 126:179 (2001)) and has been shown to synergize withTNF-alpha to prolong the survival of human neutrophils in vitro (Laan,et al. Eur Respir J 21:387 (2003)). Moreover, IL-17 is capable ofamplifying the inflammatory responses in asthma by its ability toenhance the secretion of cytokines implicated in airway remodeling suchas the profibrotic cytokines, IL-6 and IL-11 and inflammatory mediatorsgranulocyte colony-stimulating factor (G-CSF) and granulocyte macrophagecolony-stimulating factor (GM-CSF) (Molet, et al. J Allergy Clin Immunol108:430 (2001)).

Clinical evidence shows that acute, severe exacerbations of asthma areassociated with recruitment and activation of neutrophils in theairways, thus IL-17 is likely to play a significant role in asthma.Furthermore, since IL-23 is important in the maintenance anddifferentiation of IL-17 producing cells (e.g. Th17 cells), IL-23 isalso likely to play a role in asthma. Patients with mild asthma displaya detectable increase in the local concentration of free, soluble IL-17protein (Molet, et al. J Allergy Clin Immunol 108:430 (2001)) whilehealthy human volunteers with induced, severe airway inflammation due tothe exposure to a swine confinement, display a pronounced increase inthe concentration of free, soluble IL-17 protein in the bronchoalveolarspace (Fossiez et al, J Exp Med 183:2593 (1996), and Linden, et al. IntArch Allergy Immunol 126:179 (2001)). Furthermore, IL-17 levels insputum have correlated with individuals who have increased airwayhyper-reactivity Barczyk, et al. Respir Med 97:726 (2003).

In animal models of airway hyper-responsiveness, chronic inhalation ofovalbumin by sensitized mice resulted in bronchial eosinophilicinflammation and early induction of IL-17 mRNA expression in inflamedlung tissue, together with a bronchial neutrophilia Hellings, et al. AmJ Respir Cell Mol Biol 28:42 (2003). Anti-IL-17 monoclonal antibodiesstrongly reduced bronchial neutrophilic influx but significantlyenhanced IL-5 levels in both bronchoalveolar lavage fluid and serum, andaggravated allergen-induced bronchial eosinophilic influx, suggestingthat IL-17 may be involved in determining the balance between neutrophiland eosinophil accumulation following antigen insult Id.

Among the IL-17 family members, IL-17F is most closely related toIL-17A. The biological activities mediated by IL-17F are similar tothose of IL-17A, where IL-17F stimulates production of IL-6, IL-8 andG-CSF Hurst, et al. J Immunol 169:443 (2002). IL-17F also inducesproduction of IL-2, transforming growth factor (TGF)-α, and monocytechemoattractant protein (MCP) in endothelial cells Starnes, et al. JImmunol 167:4137 (2001). Similarly, allergen challenge can increaselocal IL-17F in patients with allergic asthma Kawaguchi, et al. JImmunol 167:4430 (2001). Gene delivery of IL-17F in murine lungincreases neutrophils in the bronchoalveolar space, while mucosaltransfer of the IL-17F gene enhances the levels of Ag-induced pulmonaryneutrophilia and airway responsiveness to methacholine Oda, et al. Am JRespir Crit Care Med 171:12 (2005).

Apart from asthma, several chronic inflammatory airway diseases arecharacterized by neutrophil recruitment in the airways and IL-17 hasbeen reported to play an important role in the pathogenesis ofrespiratory conditions such as chronic obstructive pulmonary disease(COPD), bacterial pneumonia and cystic fibrosis (Linden, et al. EurRespir J 15:973 (2000), Ye, et al. Am J Respir Cell Mol Biol 25:335(2001), Rahman, et al. Clin Immunol 115:268 (2005)). An anti-IL-17and/or anti-IL-23 therapeutic molecule could be demonstrated to beefficacious for chronic inflammatory airway disease in an in vitro modelof inflammation. The ability of antagonists to IL-17 and/or IL-23activity to inhibit IL-17 or and/or IL-23 signalling to induce cytokineand chemokine production from cultured HBECs or bronchial fibroblastscould be used as a measure of efficacy for such antagonists in theprevention of the production of inflammatory mediators directlyresulting from IL-17 and/or IL-23 stimulation. If the addition ofantagonists to IL-17 and/or IL-23 activity markedly reduces theproduction and expression of inflammatory mediators, it would beexpected to be efficacious in inflammatory aspects associated withchronic airway inflammation.

Multiple Sclerosis

Multiple sclerosis is a relatively commonly occurring autoimmune diseasecharacterized by demyelination and chronic inflammation of the centralnervous system (CNS). Although the mechanisms underlying diseaseinitiation are not clearly understood, the disease processes thatcontribute to clinical progression of multiple sclerosis areinflammation, demyelination, and axonal loss, or neurodegeneration.Macrophages and microglia are the main immune cells of the CNS. Thesecells, as well as T cells, neutrophils, astrocytes, and microglia, cancontribute to the immune-related pathology of, e.g., multiple sclerosis.Furthermore, T cell reactivity/autoimmunity to several myelin proteins,including myelin basic protein (MBP), proteolipid protein (PLP), myelinoligodendrocyte protein (MOG), and perhaps other myeline proteins, havebeen implicated in the induction and perpetuation of disease state andpathology of multiple sclerosis. This interaction of autoreactive Tcells and myelin proteins can result in the release of proinflammatorycytokines, including TNF-a, IFN-g, and IL-17, among others. Additionalconsequences are the proliferation of T cells, activation of B cells andmacrophages, upregulation of chemokines and adhesion molecules, and thedisruption of the blood-brain barrier. The ensuing pathology is a lossof oligodendrocytes and axons, and the formation of a demyelinated“plaque”. The plaque consists of a lesion in which the myelin sheath isnow absent and the demyelinated axons are embedded within glial scartissue. Demyelination can also occur as the result of specificrecognition and opsinization of myelin antigens by autoantibodies,followed by complement- and/or activated macrophage-mediateddestruction. It is this axonal loss and neurodegeneration that isthought to be primarily responsible for the irreversible neurologicalimpairment that is observed in progressive multiple sclerosis.

There is a large amount of clinical and pathological heterogeneity inthe course of human multiple sclerosis. Symptoms most often beginbetween the ages of 18 and 50 years old, but can begin at any age. Theclinical symptoms of multiple sclerosis can vary from mild visiondisturbances and headaches, to blindness, severe ataxia and paralysis.The majority of the patients (70-75%) have relapsing-remitting multiplesclerosis, in which disease symptoms can recur within a matter of hoursto days, followed by a much slower recovery; the absence of symptomsduring stages of remission is not uncommon. The incidence and frequencyof relapses and remissions can vary greatly, but as time progresses, therecovery phases can be incomplete and slow to occur. This worsening ofdisease in these cases is classified as secondary-progressive multiplesclerosis, and occurs in approximately 10-15% of multiple sclerosispatients. Another 10-15% of patients are diagnosed withprimary-progressive multiple sclerosis, in which disease symptoms andphysical impairment progress at a steady rate throughout the diseaseprocess.

Both IL-23 and IL-17 are overexpressed in the central nervous system ofhumans with multiple sclerosis and in mice undergoing an animal model ofmultiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Theoverexpression is observed in mice when the EAE is induced by eithermyelin oligodendrocyte glycoprotein (MOG) 35-55 peptide- or proteolipidpeptide (PLP). Furthermore, neutralization of either IL-23/p19 or IL-17results in amelioration of EAE symptoms in mice (Park et al, NatImmunol. 6:1133 (2005); Chen et al, J Clin Invest. 116:1317 (2006)).

The ability of antagonists to IL-17 and/or IL-23 activity to inhibitIL-17 or and/or IL-23 signalling-induced cytokine and chemokineproduction could be used as a measure of efficacy for such antagonistsin the treatment of multiple sclerosis. If the addition of antagoniststo IL-17 and/or IL-23 activity markedly reduces the production andexpression of inflammatory mediators (i.e. CNS-infiltrating immunecells; CNS expression of inflammatory cytokines/chemokines, etc.) andsymptoms of multiple sclerosis (e.g. paralysis; ataxia; weight loss,etc), it would be expected to be efficacious in the treatment of humans.

Cancer

Chronic inflammation has long been associated with increased incidenceof malignancy and similarities in the regulatory mechanisms have beensuggested for more than a century. Infiltration of innate immune cells,elevated activities of matrix metalloproteases (MMP) and increasedangiogenesis and vasculature density are a few examples of thesimilarities between chronic and tumour-associated inflammation.Conversely, the elimination of early malignant lesions by immunesurveillance, which relies on the cytotoxic activity oftumour-infiltrating T cells or intra-epithelial lymphocytes, is thoughtto be rate-limiting for the risk to develop cancer.

There are numerous publications describing important roles for IL-23 andIL-17 in tumor biology and/or angiogenesis. Both IL-23 and IL-17 havebeen published to be upregulated in several human tumors and cancers,including but not limited to those of the colon, breast, ovarian,cervical, prostate, lung, and stomach, as well as melanoma and T celllymphoma (Tartour et al, Cancer Res. 59:3698 (1999); Kato et al,Biochem. Biophys. Res. Commun. 282:735 (2001); Steiner et al, Prostate.56:171 (2003); Langowksi et al, Nature. May 10 [Epub ahead of print],(2006)). Thus, neutralization of both IL-17 and a key upstream regulatorof IL-17, IL-23 (via p19), is a potent and effective means of treatingcancer and other neoplastic diseases. Therefore, neutralizing both IL-17and IL-23 with antagonists of the present invention (i.e. a singleneutralizing entity or antibody to IL-17 and IL-23 or an antagonisticmolecule that will neutralize both together, such as a bispecificantibody or bispecific scFV) will have better efficacy in these diseasesthan antagonists directed toward either of IL-17 or IL-23 alone.

Angiogenesis refers to the formation of new capillaries from preexistingvessels. There are several reports that angiogenesis plays importantroles in hematological malignancies and solid tumors. The initiation ofangiogenesis and the switch to the angiogenic phenotype requires achange between proangiogenic factors and angiogenic inhibitors (Folkman,Nat. Med. 1:27 (1995)). IL-17 acts as a stimulatory hematopoieticcytokine by initiating proliferation of mature neutrophils and byexpanding myeloid progenitors. It has been well documented that IL-17has pro-angiogenic activities and stimulates the migration of vascularendothelial cells, which are associated with tumor promotion (Numasakiet al, Blood, 101:2620 (2003); Yang et al, J. Biol. Chem., 278:33232(2003); Fujino et al, Gut, 52:65 (2003)). In vitro angiogenic activitycan be suppressed by neutralizing IL-17 with a neutralizing anti-IL-17monoclonal antibody, further supporting the role of IL-17 in thisaction. It is also able to selectively enhance mitogenic activity ofbasic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF),and vascular endothelial growth factor (VEGF), and IL-17 may alsopromote bFGF-, HGF- and VEGF-mediated angiogenesis via bFGF-, HGF- andVEGF-induced growth of vascular endothelial cells (Takahashi et al,Immunol Lett. 98:189 (2005)). IL-17 has been reported to augment thesecretion of several angiogenic CXC chemokines (e.g. CXCL1, CXCL5,CXCL6, and CXCL8) in non-small cell lung cancer (NSCLC) lines.Endothelial cell chemotactic activity (a measure of net angiogenicpotential) is increased in response to conditioned medium from NSCLCstimulated with recombinant IL-17. NSCLC lines transfected with IL-17grew more rapidly versus controls when transplanted in SCID mice(Numasaki et al, J Immunol. 175:6177 (2005)). Furthermore, IL-17 hasbeen reported to be associated with increased IL-6 at the site of tumorsand is well reported to increase MMP-9 expression. MMP-9 is an importantmodulator in diseases of inflammation, autoimmunity, and cancer. Thesereports, therefore, clearly implicate a pro-angiogenic and tumorpromoting action for IL-17. Therefore, neutralizing both IL-17 and IL23with antagonists of the present invention (i.e. a single neutralizingentity or antibody to IL-17 and IL-23 or an antagonistic molecule thatwill neutralize both together, such as a bispecific antibody orbispecific scFV) will have better efficacy than antagonists directedtoward either of IL-17 or IL-23 alone.

Similar to IL-17, IL-23 promotes inflammatory responses includingupregulation of MMP9, and is also reported to increase angiogenesis andreduce CD8+ T-cell infiltration. Taken together, these actions can leadto enhanced initiation, progression, and/or maintenance of tumors,cancers, and other transformed growths. That IL-23 plays an importantrole in cancerous diseases is supported by the observation thatneutralization of IL-23 with a monoclonal antibody or with geneticdeletion in mice reduces tumor growth in several murine tumor models(Langowksi et al. Nature. May 10 (2006) [Epub ahead of print]). Efficacyis associated with reduced IL-17 expression and reductions inIL-17-related tumorogenic biomarkers, such as granulocyte infiltration,G-CSF and MMP-9. Therefore, neutralizing both IL-17 and IL23 withantagonists of the present invention (i.e. a single neutralizing entityor antibody to IL-17 and IL-23 or an antagonistic molecule that willneutralize both together, such as a bispecific antibody or bispecificscFV) will have better efficacy in these diseases than antagonistsdirected toward either of IL-17 or IL-23 alone.

Irritable Bowel Syndrome (“IBS”)

Irritable bowel syndrome represents a disease characterized by abdominalpain or discomfort and an erratic bowel habit. IBS patients can becharacterized into three main groups based on bowel habits: those withpredominantly loose or frequent stools, those with predominantly hard orinfrequent stools, and those with variable or normal stools (Talley etal., 2002). Altered intestinal motility, abnormalities in epithelialfunction, abnormal transit of stool and gas, and stress, may contributeto symptoms, while visceral hypersensitivity is a key feature in mostpatients. Genetic factors affecting pain-signaling and disturbances incentral processing of afferent signals are postulated to predisposeindividuals to IBS following specific environmental exposures. Studieshave also demonstrated that inflammatory responses in the colon maycontribute to increased sensitivity of smooth muscle and enteric nervesand therefore perturb sensory-motor functions in the intestine (Collinset al., 2001). There is clinical overlap between IBS and IBD, withIBS-like symptoms frequently reported in patients before the diagnosisof IBD, and a higher than expected IBS symptoms in patients in remissionfrom established IBD. Thus, these conditions may coexist with a higherthan expected frequency, or may exist on a continuum, with IBS and IBDat different ends of the same spectrum. However, it should be noted thatin most IBS patients, colonic biopsy specimens appear normal.Nevertheless, IBS significantly affects a very large number ofindividuals (U.S. prevalence in 2000, approximately 16 millionindividuals), resulting in a total cost burden of 1.7 billion dollars(year 2000). Thus, among the most prevalent and costly gastrointestinaldiseases and disorders, IBS is second only to gastroesophageal refluxdisease (GERD). Yet unlike GERD, treatment for IBS remainsunsatisfactory (Talley et al., 2002; Farhadi et al., 21001; Collins etal., 2001), demonstrating that IBS clearly represents an unmet medicalneed.

Converging disease models have been proposed that postulate an enhancedresponsiveness of neural, immune or neuroimmune circuits in the centralnervous system (CNS) or in the gut to central (psychosocial) orperipheral (tissue irritation, inflammation, infection) perturbations ofnormal homeostasis (Talley et al., 2002). This enhanced responsivenessresults in dysregulation of gut motility, epithelial function (immune,permeability), and visceral hypersensitivity, which in turn results inIBS symptoms.

There may be a role for a number of different molecules in thepathogenesis of IBS including a role for molecules that stimulateneurons and those that are involved in initiation of inflammatoryprocess. A number of our in-house molecules are known to be linked topossible activity on neurons due to their direct expression by neuronsor expression of their receptors on neurons, including IL-17D, IL-17Band IL-31. Moreover, a number of IL-17 family members and relatedmolecules have been associated with inflammation in the gut, includingIL-17A, IL-17F, IL-23 and IL-31.

Efficacy of inhibitors of these molecules could be tested in vivo inanimal models of disease. Several animal models have been proposed thatmimic key features of IBS and involve centrally targeted stimuli(stress) or peripherally targeted stimuli (infection, inflammation). Twoexamples of in vivo animal models that can be used to determine theeffectiveness of inhibitors in the treatment of IBS are (i) modelsfocusing on primary CNS-directed pathogeneisis of IBS (stress models),and (ii) models focusing on gut-directed inducers of stress (i.e. gutinflammation, infection or physical stress). It should be noted however,that events within the CNS or in the gastrointestinal (GI) tract do notoccur in isolation and that symptoms of IBS most likely result from acomplex interaction between signals from the CNS on the GI and viceversa.

Thus, in summary, there are several molecules and pathogenic pathwaysthat are shared by IL-17 and IL-23 which play important roles in thedevelopment, progression, and maintenance of both autoimmune diseasesand cancerous diseases. These include the pro-angiogenic roles of IL-17and IL-23; enhanced MMP-9 levels and activity by IL-17 and IL-23; IL-23,TGF-b and IL-6-mediated production and/or maintenece of Th17 cells;roles of TGF-b and IL-6 in the generation of Foxp3+ regulatory T cells;and additional pathways and molecules. Therefore, the IL-17/IL-23 axisrepresents an important link to the inappropriate and pathogenic T cellresponses associated with autoimmune diseases, tumour-promotingpro-inflammatory processes, and the failure of the adaptive immunesurveillance to infiltrate tumours. Therefore, neutralizing both IL-17and IL23 with antagonists of the present invention (i.e. a singleneutralizing entity or antibody to IL-17 and IL-23 or an antagonisticmolecule that will neutralize both together, such as a bispecificantibody or bispecific scFV) will have better efficacy in these diseasesthan antagonists directed toward either of IL-17 or IL-23 alone.

For pharmaceutical use, the antibodies of the present invention areformulated for parenteral, particularly intravenous or subcutaneous,delivery according to conventional methods. Intravenous administrationwill be by bolus injection, controlled release, e.g, using mini-pumps orother appropriate technology, or by infusion over a typical period ofone to several hours. In general, pharmaceutical formulations willinclude a hematopoietic protein in combination with a pharmaceuticallyacceptable vehicle, such as saline, buffered saline, 5% dextrose inwater or the like. Formulations may further include one or moreexcipients, preservatives, solubilizers, buffering agents, albumin toprevent protein loss on vial surfaces, etc. When utilizing such acombination therapy, the cytokines may be combined in a singleformulation or may be administered in separate formulations. Methods offormulation are well known in the art and are disclosed, for example, inRemington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co.,Easton Pa., 1990, which is incorporated herein by reference. Therapeuticdoses will generally be in the range of 0.1 to 100 mg/kg of patientweight per day, preferably 0.5-20 mg/kg per day, with the exact dosedetermined by the clinician according to accepted standards, taking intoaccount the nature and severity of the condition to be treated, patienttraits, etc. Determination of dose is within the level of ordinary skillin the art. More commonly, the proteins will be administered over oneweek or less, often over a period of one to three days. In general, atherapeutically effective amount of antibodies of the present inventionis an amount sufficient to produce a clinically significant increase inthe proliferation and/or differentiation of lymphoid or myeloidprogenitor cells, which will be manifested as an increase in circulatinglevels of mature cells (e.g. platelets or neutrophils). The antibodiesof the present invention can also be administered in combination withother cytokines such as IL-3, -6 and -11; stem cell factor;erythropoietin; G-CSF and GM-CSF. Within regimens of combinationtherapy, daily doses of other cytokines are commonly known by oneskilled in the art, or can be determined without undue experimentation.Combination therapy with EPO, for example, is indicated in anemicpatients with low EPO levels.

Generally, the dosage of administered antibodies will vary dependingupon such factors as the patient's age, weight, height, sex, generalmedical condition and previous medical history. Typically, it isdesirable to provide the recipient with a dosage of antibodies which isin the range of from about 1 pg/kg to 10 mg/kg (amount of agent/bodyweight of patient), although a lower or higher dosage also may beadministered as circumstances dictate.

Administration of antibodies of the invention to a subject can beintravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, intrapleural, intrathecal, by perfusion through a regionalcatheter, or by direct intralesional injection. When administeringtherapeutic proteins by injection, the administration may be bycontinuous infusion or by single or multiple boluses.

Additional routes of administration include oral, mucosal-membrane,pulmonary, and transcutaneous. Oral delivery is suitable for polyestermicrospheres, zein microspheres, proteinoid microspheres,polycyanoacrylate microspheres, and lipid-based systems (see, forexample, DiBase and Morrel, “Oral Delivery of MicroencapsulatedProteins,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 255-288 (Plenum Press 1997)). The feasibility of anintranasal delivery is exemplified by such a mode of insulinadministration (see, for example, Hinchcliffe and Illum, Adv. DrugDeliv. Rev. 35:199 (1999)). Dry or liquid particles comprisingantibodies of the invention can be prepared and inhaled with the aid ofdry-powder dispersers, liquid aerosol generators, or nebulizers (e.g.,Pettit and Gombotz, TIBTECH 16:343 (1998); Patton et al., Adv. DrugDeliv. Rev. 35:235 (1999)). This approach is illustrated by the AERXdiabetes management system, which is a hand-held electronic inhaler thatdelivers aerosolized insulin into the lungs. Studies have shown thatproteins as large as 48,000 kDa have been delivered across skin attherapeutic concentrations with the aid of low-frequency ultrasound,which illustrates the feasibility of transcutaneous administration(Mitragotri et al., Science 269:850 (1995)). Transdermal delivery usingelectroporation provides another means to administer a molecule havingIL-17 and IL-23/p19 binding activity (Potts et al., Pharm. Biotechnol.10:213 (1997)).

A pharmaceutical composition comprising an antibodies of the inventioncan be formulated according to known methods to prepare pharmaceuticallyuseful compositions, whereby the therapeutic proteins are combined in amixture with a pharmaceutically acceptable carrier. A composition issaid to be a “pharmaceutically acceptable carrier” if its administrationcan be tolerated by a recipient patient. Sterile phosphate-bufferedsaline is one example of a pharmaceutically acceptable carrier. Othersuitable carriers are well-known to those in the art. See, for example,Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (MackPublishing Company 1995).

For purposes of therapy, antibodies of the invention and apharmaceutically acceptable carrier are administered to a patient in atherapeutically effective amount. A combination of a therapeuticmolecule of the present invention and a pharmaceutically acceptablecarrier is said to be administered in a “therapeutically effectiveamount” if the amount administered is physiologically significant. Anagent is physiologically significant if its presence results in adetectable change in the physiology of a recipient patient. For example,an agent used to treat inflammation is physiologically significant ifits presence alleviates the inflammatory response. Effective treatmentmay be assessed in a variety of ways. In one embodiment, effectivetreatment is determined by reduced inflammation. In other embodiments,effective treatment is marked by inhibition of inflammation. In stillother embodiments, effective therapy is measured by increased well-beingof the patient including such signs as weight gain, regained strength,decreased pain, thriving, and subjective indications from the patient ofbetter health.

A pharmaceutical composition comprising antibodies of the invention canbe furnished in liquid form, in an aerosol, or in solid form. Liquidforms, are illustrated by injectable solutions and oral suspensions.Exemplary solid forms include capsules, tablets, and controlled-releaseforms. The latter form is illustrated by miniosmotic pumps and implants(Bremer et al., Pharm. Biotechnol. 10:239 (1997); Ranade, “Implants inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 95-123 (CRC Press 1995); Bremer et al., “Protein Delivery withInfusion Pumps,” in Protein Delivery: Physical Systems, Sanders andHendren (eds.), pages 239-254 (Plenum Press 1997); Yewey et al.,“Delivery of Proteins from a Controlled Release Injectable Implant,” inProtein Delivery: Physical Systems, Sanders and Hendren (eds.), pages93-117 (Plenum Press 1997)).

Liposomes provide one means to deliver therapeutic polypeptides to asubject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.Infect. Dis. 12 (Suppl. 1):561 (1993), Kim, Drugs 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers,” inDrug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRCPress 1995)). Liposomes are similar in composition to cellular membranesand as a result, liposomes can be administered safely and arebiodegradable. Depending on the method of preparation, liposomes may beunilamellar or multilamellar, and liposomes can vary in size withdiameters ranging from 0.02 μm to greater than 10 μm. A variety ofagents can be encapsulated in liposomes: hydrophobic agents partition inthe bilayers and hydrophilic agents partition within the inner aqueousspace(s) (see, for example, Machy et al., Liposomes In Cell Biology AndPharmacology (John Libbey 1987), and Ostro et al., American J. Hosp.Pharm. 46:1576 (1989)). Moreover, it is possible to control thetherapeutic availability of the encapsulated agent by varying liposomesize, the number of bilayers, lipid composition, as well as the chargeand surface characteristics of the liposomes.

Liposomes can adsorb to virtually any type of cell and then slowlyrelease the encapsulated agent. Alternatively, an absorbed liposome maybe endocytosed by cells that are phagocytic. Endocytosis is followed byintralysosomal degradation of liposomal lipids and release of theencapsulated agents (Scherphof et al., Ann. N.Y. Acad Sci. 446:368(1985)). After intravenous administration, small liposomes (0.1 to 1.0μm) are typically taken up by cells of the reticuloendothelial system,located principally in the liver and spleen, whereas liposomes largerthan 3.0 μm are deposited in the lung. This preferential uptake ofsmaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

The reticuloendothelial system can be circumvented by several methodsincluding saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (Claassen etal., Biochim. Biophys. Acta 802:428 (1984)). In addition, incorporationof glycolipid- or polyethelene glycol-derivatized phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system (Allen et al., Biochim.Biophys. Acta 1068:133 (1991); Allen et al., Biochim. Biophys. Acta1150:9 (1993)).

Liposomes can also be prepared to target particular cells or organs byvarying phospholipid composition or by inserting receptors or ligandsinto the liposomes. For example, liposomes, prepared with a high contentof a nonionic surfactant, have been used to target the liver (Hayakawaet al., Japanese Patent 04-244,018; Kato et al., Biol. Pharm. Bull.16:960 (1993)). These formulations were prepared by mixing soybeanphosphatidylcholine, α-tocopherol, and ethoxylated hydrogenated castoroil (HCO-60) in methanol, concentrating the mixture under vacuum, andthen reconstituting the mixture with water. A liposomal formulation ofdipalmitoylphosphatidylcholine (DPPC) with a soybean-derivedsterylglucoside mixture (SG) and cholesterol (Ch) has also been shown totarget the liver (Shimizu et al., Biol. Pharm. Bull. 20:881 (1997)).

Alternatively, various targeting ligands can be bound to the surface ofthe liposome, such as antibodies, antibody fragments, carbohydrates,vitamins, and transport proteins. For example, liposomes can be modifiedwith branched type galactosyllipid derivatives to targetasialoglycoprotein (galactose) receptors, which are exclusivelyexpressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev.Ther. Drug Carrier Syst. 14:287 (1997); Murahashi et al., Biol. Pharm.Bull. 20:259 (1997)). Similarly, Wu et al., Hepatology 27:772 (1998),have shown that labeling liposomes with asialofetuin led to a shortenedliposome plasma half-life and greatly enhanced uptake ofasialofetuin-labeled liposome by hepatocytes. On the other hand, hepaticaccumulation of liposomes comprising branched type galactosyllipidderivatives can be inhibited by preinjection of asialofetuin (Murahashiet al., Biol. Pharm. Bull. 20:259 (1997)). Polyaconitylated human serumalbumin liposomes provide another approach for targeting liposomes toliver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681 (1997)).Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe ahepatocyte-directed liposome vesicle delivery system, which hasspecificity for hepatobiliary receptors associated with the specializedmetabolic cells of the liver.

In a more general approach to tissue targeting, target cells areprelabeled with biotinylated antibodies specific for a ligand expressedby the target cell (Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).After plasma elimination of free antibody, streptavidin-conjugatedliposomes are administered. In another approach, targeting antibodiesare directly attached to liposomes (Harasym et al., Adv. Drug Deliv.Rev. 32:99 (1998)).

Antibodies can be encapsulated within liposomes using standardtechniques of protein microencapsulation (see, for example, Anderson etal., Infect. Immun. 31:1099 (1981), Anderson et al., Cancer Res. 50:1853(1990), and Cohen et al., Biochim. Biophys. Acta 1063:95 (1991), Alvinget al. “Preparation and Use of Liposomes in Immunological Studies,” inLiposome Technology, 2nd Edition, Vol. III, Gregoriadis (ed.), page 317(CRC Press 1993), Wassef et al., Meth. Enzymol. 149:124 (1987)). Asnoted above, therapeutically useful liposomes may contain a variety ofcomponents. For example, liposomes may comprise lipid derivatives ofpoly(ethylene glycol) (Allen et al., Biochim. Biophys. Acta 1150:9(1993)).

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332 (1995); Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney andBurke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin. Chem.Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres canalso provide carriers for intravenous administration of therapeuticproteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167(1997)).

The present invention also contemplates chemically modified polypeptideshaving binding IL-17 and IL-23 activity such as anti-IL-17A andIL-23/p19 antibodies, which a polypeptide is linked with a polymer, asdiscussed above.

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19^(th) Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

As an illustration, pharmaceutical compositions may be supplied as a kitcomprising a container that comprises an antibody of the invention.Antibodies of the invention can be provided in the form of an injectablesolution for single or multiple doses, or as a sterile powder that willbe reconstituted before injection. Alternatively, such a kit can includea dry-powder disperser, liquid aerosol generator, or nebulizer foradministration of a therapeutic polypeptide. Such a kit may furthercomprise written information on indications and usage of thepharmaceutical composition. Moreover, such information may include astatement that the antibody composition is contraindicated in patientswith known hypersensitivity to IL-17 and IL-23.

A pharmaceutical composition comprising antibodies of the invention canbe furnished in liquid form, in an aerosol, or in solid form. Liquidforms, are illustrated by injectable solutions, aerosols, droplets,topological solutions and oral suspensions. Exemplary solid formsinclude capsules, tablets, and controlled-release forms. The latter formis illustrated by miniosmotic pumps and implants (Bremer et al., Pharm.Biotechnol. 10:239 (1997); Ranade, “Implants in Drug Delivery,” in DrugDelivery Systems, Ranade and Hollinger (eds.), pages 95-123 (CRC Press1995); Bremer et al., “Protein Delivery with Infusion Pumps,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 239-254(Plenum Press 1997); Yewey et al., “Delivery of Proteins from aControlled Release Injectable Implant,” in Protein Delivery: PhysicalSystems, Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)).Other solid forms include creams, pastes, other topologicalapplications, and the like.

Liposomes provide one means to deliver therapeutic polypeptides to asubject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.Infect. Dis. 12 (Suppl. 1):S61 (1993), Kim, Drugs 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers,” inDrug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRCPress 1995)). Liposomes are similar in composition to cellular membranesand as a result, liposomes can be administered safely and arebiodegradable. Depending on the method of preparation, liposomes may beunilamellar or multilamellar, and liposomes can vary in size withdiameters ranging from 0.02 μm to greater than 10 μm. A variety ofagents can be encapsulated in liposomes: hydrophobic agents partition inthe bilayers and hydrophilic agents partition within the inner aqueousspace(s) (see, for example, Machy et al., Liposomes In Cell Biology AndPharmacology (John Libbey 1987), and Ostro et al., American J. Hosp.Pharm. 46:1576 (1989)). Moreover, it is possible to control thetherapeutic availability of the encapsulated agent by varying liposomesize, the number of bilayers, lipid composition, as well as the chargeand surface characteristics of the liposomes.

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19^(th) Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

The present invention contemplates antagonists of IL-17 and IL-23 andmethods and therapeutic uses comprising an such antagonists as describedherein. Such compositions can further comprise a carrier. The carriercan be a conventional organic or inorganic carrier. Examples of carriersinclude water, buffer solution, alcohol, propylene glycol, macrogol,sesame oil, corn oil, and the like.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Antibodies that Bind IL-17A and IL-17F

Hybridomas expressing monoclonal antibodies to IL-17A and IL-17F weredescribed in co-pending and co-owned US Patent Publication No.2007-0218065, published Sep. 20, 2007 and in U.S. patent applicationSer. No. 11/741,189, filed Apr. 27, 2007, herein incorporated byreference. These hybridomas were deposited with the American Type TissueCulture Collection (ATCC; 10801 University Blvd, Manassas Va.20110-2209) patent depository as original deposits under the BudapestTreaty and were given the following ATCC Accession Nos.: clone339.15.5.3 (ATCC Patent Deposit Designation PTA-7987, deposited on Nov.7, 2006); clone 339.15.3.6 (ATCC Patent Deposit Designation PTA-7988,deposited on Nov. 7, 2006); and clone 339.15.6.16 (ATCC Patent DepositDesignation PTA-7989, deposited on Nov. 7, 2006. The variable heavyregions and variable light regions of the antibodies expressed by thesehybridomas can be determined by amino acid sequencing. The polypeptidescomprising the variable heavy regions or variable light regions can alsobe separated by conventional protein isolation techniques. Thecomplementarity determining regions (CDRs) of the heavy and lightvariable regions can be determined by one of ordinary skill in the art.Thus, heavy chain and light chain complementarity determining regionsand variable heavy and light regions can be expressed in cell cultureand purified or produced synthetically.

Example 2 Antibodies that Bind the p-19 Subunit of IL-23

Antibodies and antibody fragments that bind to IL-23p19 were identifiedby screening a phage display library designed so that the antibodylight-chain variable region and a portion of the heavy-chain variableregion are combined with synthetic DNA encoding human antibodysequences, which are then displayed on phage and phagemid libraries asFab antibody fragments (Dyax® Human Antibody Libraries, Dyax Corp.,Cambridge, Mass.). These antibodies and antibody fragments and aredescribed in co-pending and co-owned U.S. patent application Ser. No.11/762,738, filed Jun. 13, 2007 and WIPO Publication Number 2007/147019,published Dec. 21, 2007, herein incorporated by reference. The aminoacid sequences of the variable heavy and variable light regions of thesesequences are shown in Table 1, below.

The variable light and heavy chain fragments of antibodies can beisolated in a Fab format. These variable regions can then be manipulatedto generate antibodies, including antigen-binding fragments, such asscFvs, to IL-23p19. Using this technology the variable regions of Fabshave been identified for their characteristics of binding and orneutralizing IL-23p19 in plate-based assays described in WIPOPublication Number 2007/147019.

TABLE 1 VL poly- VH pep- poly- tide peptide Clus- SEQ SEQ Light LightLight Light Light Light Light Heavy Heavy Heavy Heavy Heavy Heavy Heavyter ID ID FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4# NO: NO: range range range range range range range range range rangerange range range range 26 7 8 1-23 24-35 36-50 51-57 58-89 90-98 99-108 1-30 31-35 36-49 50-66 67-98 99-105 106-116 27 9 10 1-23 24-3435-49 50-56 57-88 89-97  98-107 1-30 31-35 36-49 50-66 67-98 99-106107-117 28 11 12 1-23 24-35 36-50 51-57 58-89 90-98  99-108 1-30 31-3536-49 50-66 67-98 99-108 109-119 29 13 14 1-22 23-36 37-51 52-58 59-90 91-102 103-112 1-30 31-35 36-49 50-66 67-98 99-112 113-123 33 15 161-23 24-34 35-49 50-56 57-88 89-97  98-107 1-30 31-35 36-49 50-66 67-9899-113 114-124 36 17 18 1-23 24-34 35-49 50-56 57-88 89-97  98-107 1-3031-35 36-49 50-66 67-98 99-105 106-116 40 19 20 1-22 23-33 34-48 49-5556-87 88-96  97-106 1-30 31-35 36-49 50-66 67-98 99-116 117-127 41 21 221-23 24-39 40-54 55-61 62-93  94-102 103-112 1-30 31-35 36-49 50-6667-98 99-112 113-123 43 23 24 1-23 24-39 40-54 55-61 62-93  94-102103-112 1-30 31-35 36-49 50-66 67-98 99-107 108-118 101 25 26 1-22 23-3637-51 52-58 59-90  91-102 103-112 1-30 31-35 36-49 50-66 67-98 99-112113-123 102 27 28 1-23 24-34 35-49 50-56 57-88 89-97  98-107 1-30 31-3536-49 50-66 67-98 99-110 111-121 103 29 30 1-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-35 36-49 50-66 67-98 99-113 114-124 110 31 321-22 23-35 36-50 51-57 58-89 90-98  99-108 1-30 31-35 36-49 50-66 67-9899-119 120-130 114 33 34 1-23 24-34 35-49 50-56 57-88 89-97  98-107 1-3031-35 36-49 50-66 67-98 99-108 109-119 115 35 36 1-23 24-39 40-54 55-6162-93  94-102 103-112 1-30 31-35 36-49 50-66 67-98 99-108 109-119 119 3738 1-23 24-34 35-49 50-56 57-88 89-97  98-107 1-30 31-35 36-49 50-6667-98 99-106 107-117 120 39 40 1-23 24-39 40-54 55-61 62-93  94-102103-112 1-30 31-35 36-49 50-66 67-98 99-108 109-119 121 41 42 1-23 24-3435-49 50-56 57-88 89-97  98-107 1-30 31-35 36-49 50-66 67-98 99-109110-120 122 43 44 1-23 24-34 35-49 50-56 57-88 89-96  97-106 1-30 31-3536-49 50-66 67-98 99-108 109-119 123 45 46 1-22 23-33 34-48 49-55 56-8788-97  98-107 1-30 31-35 36-49 50-66 67-98 99-111 112-122 124 47 48 1-2324-34 35-49 50-56 57-88 89-97  98-107 1-30 31-35 36-49 50-66 67-9899-110 111-121 125 49 50 1-23 24-34 35-49 50-56 57-88 89-97  98-107 1-3031-35 36-49 50-66 67-98 99-106 107-117 126 51 52 1-22 23-35 36-50 51-5758-89  90-100 101-110 1-30 31-35 36-49 50-66 67-98 99-106 107-117 127 5354 1-22 23-35 36-50 51-57 58-89  90-100 101-110 1-30 31-35 36-49 50-6667-98 99-111 112-122 128 55 56 1-22 23-35 36-50 51-57 58-89  90-100101-110 1-30 31-35 36-49 50-66 67-98 99-111 112-122 129 57 58 1-23 24-3435-49 50-56 57-88 89-98  99-108 1-30 31-35 36-49 50-66 67-98 99-107108-118 130 59 60 1-23 24-34 35-49 50-56 57-88 89-97  98-107 1-30 31-3536-49 50-66 67-98 99-109 110-120 131 61 62 1-23 24-34 35-49 50-56 57-8889-97  98-107 1-30 31-35 36-49 50-66 67-98 99-109 110-120 132 63 64 1-2324-34 35-49 50-56 57-88 89-97  98-107 1-30 31-35 36-49 50-66 67-9899-115 116-126 134 65 66 1-22 23-33 34-48 49-55 56-87 88-96  97-106 1-3031-35 36-49 50-66 67-98 99-111 112-122 135 67 68 1-23 24-34 35-49 50-5657-88 89-98  99-108 1-30 31-35 36-49 50-66 67-98 99-116 117-127 136 6970 1-23 24-34 35-49 50-56 57-88 89-98  99-108 1-30 31-35 36-49 50-6667-98 99-109 110-120 137 71 72 1-22 23-36 37-51 52-58 59-90  91-100101-110 1-30 31-35 36-49 50-66 67-98 99-105 106-116 138 73 74 1-23 24-3940-54 55-61 62-93  94-102 103-112 1-30 31-35 36-49 50-66 67-98 99-107108-118 139 75 76 1-23 24-34 35-49 50-56 57-88 89-97  98-107 1-30 31-3536-49 50-66 67-98 99-111 112-122 140 77 78 1-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-35 36-49 50-66 67-98 99-111 112-122 141 79 801-23 24-39 40-54 55-61 62-93  94-102 103-112 1-30 31-35 36-49 50-6667-98 99-107 108-118 142 81 82 1-23 24-34 35-49 50-56 57-88 89-97 98-107 1-30 31-35 36-49 50-66 67-98 99-113 114-124 143 83 84 1-23 24-3435-49 50-56 57-88 89-97  98-107 1-30 31-35 36-49 50-66 67-98 99-107108-118 144 85 86 1-23 24-39 40-54 55-61 62-93  94-102 103-112 1-3031-35 36-49 50-66 67-98 99-111 112-122 145 87 88 1-23 24-34 35-49 50-5657-88 89-96  97-106 1-30 31-35 36-49 50-66 67-98 99-107 108-118 146 8990 1-22 23-36 37-51 52-58 59-90  91-100 101-110 1-30 31-35 36-49 50-6667-98 99-111 112-122 148 91 92 1-23 24-34 35-49 50-56 57-88 89-97 98-107 1-30 31-35 36-49 50-66 67-98 99-105 106-116 149 93 94 1-23 24-3435-49 50-56 57-88 89-98  99-108 1-30 31-35 36-49 50-66 67-98 99-105106-116 150 95 96 1-23 24-39 40-54 55-61 62-93  94-101 102-111 1-3031-35 36-49 50-66 67-98 99-108 109-119 151 97 98 1-23 24-34 35-49 50-5657-88 89-97  98-107 1-30 31-35 36-49 50-66 67-98 99-108 109-119 152 99100 1-23 24-34 35-49 50-56 57-88 89-98  99-108 1-30 31-35 36-49 50-6566-97 98-110 111-121 153 101 102 1-23 24-34 35-49 50-56 57-88 89-97 98-107 1-30 31-35 36-49 50-66 67-98 99-110 111-121 154 103 104 1-2324-39 40-54 55-61 62-93  94-102 103-112 1-30 31-35 36-49 50-66 67-9899-107 108-118 155 105 106 1-23 24-35 36-50 51-57 58-89  90-100 101-1101-30 31-35 36-49 50-66 67-98 99-109 110-120 156 107 108 1-22 23-35 36-5051-57 58-89  90-100 101-110 1-30 31-35 36-49 50-66 67-98 99-109 110-120157 109 110 1-22 23-35 36-50 51-57 58-89  90-100 101-110 1-30 31-3536-49 50-66 67-98 99-113 114-124 158 111 112 1-23 24-39 40-54 55-6162-93  94-102 103-112 1-30 31-35 36-49 50-66 67-98 99-113 114-124 159113 114 1-23 24-39 40-54 55-61 62-93  94-102 103-112 1-30 31-35 36-4950-66 67-98 99-112 113-123 160 115 116 1-23 24-34 35-49 50-56 57-8889-96  97-106 1-30 31-35 36-49 50-66 67-98 99-105 106-116 161 117 1181-23 24-39 40-54 55-61 62-93  94-102 103-112 1-30 31-35 36-49 50-6667-98 99-107 108-118 162 119 120 1-23 24-35 36-50 51-57 58-89 90-98 99-108 1-30 31-35 36-49 50-66 67-98 99-105 106-116 163 121 122 1-2324-35 36-50 51-57 58-89 90-98  99-108 1-30 31-35 36-49 50-66 67-9899-108 109-119 164 123 124 1-23 24-35 36-50 51-57 58-89 90-98  99-1081-30 31-35 36-49 50-66 67-98 99-107 108-118 165 125 126 1-23 24-39 40-5455-61 62-93  94-102 103-112 1-30 31-35 36-49 50-66 67-98 99-109 110-120166 127 128 1-23 24-35 36-50 51-57 58-89 90-98  99-108 1-30 31-35 36-4950-66 67-98 99-105 106-116 167 129 130 1-23 24-34 35-49 50-56 57-8889-97  98-107 1-30 31-35 36-49 50-66 67-98 99-103 104-114 168 131 1321-23 24-34 35-49 50-56 57-88 89-97  98-107 1-30 31-35 36-49 50-66 67-9899-108 109-119 169 133 134 1-23 24-35 36-50 51-57 58-89 90-99 100-1091-30 31-35 36-49 50-66 67-98 99-112 113-123 170 135 136 1-23 24-39 40-5455-61 62-93  94-101 102-111 1-30 31-35 36-49 50-66 67-98 99-112 113-123171 137 138 1-23 24-35 36-50 51-57 58-89  90-101 102-111 1-30 31-3536-49 50-66 67-98 99-112 113-123 172 139 140 1-23 24-34 35-49 50-5657-88 89-96  97-106 1-30 31-35 36-49 50-66 67-98 99-107 108-118 173 141142 1-23 24-34 35-49 50-56 57-88 89-97  98-107 1-30 31-35 36-49 50-6667-98 99-109 110-120 174 143 144 1-23 24-34 35-49 50-56 57-88 89-97 98-107 1-30 31-35 36-49 50-66 67-98 99-106 107-117 175 145 146 1-2324-34 35-49 50-56 57-88 89-99 100-109 1-30 31-35 36-49 50-66 67-9899-112 113-123 176 147 148 1-23 24-34 35-49 50-56 57-88 89-98  99-1081-30 31-35 36-49 50-66 67-98 99-109 110-120 178 149 150 1-23 24-39 40-5455-61 62-93  94-101 102-111 1-30 31-35 36-49 50-66 67-98 99-113 114-124179 151 152 1-23 24-39 40-54 55-61 62-93  94-102 103-112 1-30 31-3536-49 50-66 67-98 99-110 111-121 180 153 154 1-23 24-34 35-49 50-5657-88 89-98  99-108 1-30 31-35 36-49 50-66 67-98 99-109 110-120 181 155156 1-23 24-34 35-49 50-56 57-88 89-97  98-107 1-30 31-35 36-49 50-6667-98 99-114 115-125 182 157 158 1-23 24-39 40-54 55-61 62-93  94-102103-112 1-30 31-35 36-49 50-66 67-98 99-108 109-119 183 159 160 1-2324-35 36-50 51-57 58-89 90-98  99-108 1-30 31-35 36-49 50-66 67-9899-107 108-118 184 161 162 1-23 24-34 35-49 50-56 57-88 89-97  98-1071-30 31-35 36-49 50-66 67-98 99-117 118-128 185 163 164 1-23 24-39 40-5455-61 62-93  94-101 102-111 1-30 31-35 36-49 50-66 67-98 99-110 111-121186 165 166 1-23 24-35 36-50 51-57 58-89 90-99 100-109 1-30 31-35 36-4950-66 67-98 99-107 108-118 187 167 168 1-23 24-34 35-49 50-56 57-8889-97  98-107 1-30 31-35 36-49 50-66 67-98 99-107 108-118 188 169 1701-23 24-39 40-54 55-61 62-93  94-103 104-113 1-30 31-35 36-49 50-6667-98 99-111 112-122 189 171 172 1-23 24-39 40-54 55-61 62-93  94-102103-112 1-30 31-35 36-49 50-66 67-98 99-109 110-120 190 173 174 1-2324-39 40-54 55-61 62-93  94-102 103-112 1-30 31-35 36-49 50-66 67-9899-105 106-116 194 175 176 1-23 24-39 40-54 55-61 62-93  94-102 103-1121-30 31-35 36-49 50-66 67-98 99-113 114-124 197 177 178 1-23 24-39 40-5455-61 62-93  94-102 103-112 1-30 31-35 36-49 50-66 67-98 99-113 114-124198 179 180 1-23 24-39 40-54 55-61 62-93  94-102 103-112 1-30 31-3536-49 50-66 67-98 99-106 107-117 201 181 182 1-23 24-39 40-54 55-6162-93  94-102 103-112 1-30 31-35 36-49 50-66 67-98 99-110 111-121 205183 184 1-23 24-39 40-54 55-61 62-93  94-102 103-112 1-30 31-35 36-4950-66 67-98 99-113 114-124 206 185 186 1-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-35 36-49 50-66 67-98 99-112 113-123 208 187 — —— — — — — 1-30 31-35 36-49 50-66 67-98 99-112 113-123 211 188 189 1-2324-39 40-54 55-61 62-93  94-102 103-112 1-30 31-35 36-49 50-66 67-9899-110 111-121 251 190 191 1-22 23-33 34-48 49-55 56-87 88-97  98-1071-30 31-35 36-49 50-66 67-98 99-115 116-126 252 192 193 1-22 23-33 34-4849-55 56-87 88-96  97-106 1-30 31-35 36-49 50-66 67-98 99-120 121-131253 194 195 1-22 23-33 34-48 49-55 56-87 88-96  97-106 1-30 31-35 36-4950-66 67-98 99-115 116-126 254 196 197 1-22 23-33 34-48 49-55 56-8788-96  97-106 1-30 31-35 36-49 50-66 67-98 99-115 116-126 255 198 1991-23 24-35 36-50 51-57 58-89 90-98  99-108 1-30 31-35 36-49 50-66 67-9899-114 115-125 256 200 201 1-22 23-33 34-48 49-55 56-87 88-96  97-1061-30 31-35 36-49 50-66 67-98 99-105 106-116 257 202 203 1-22 23-33 34-4849-55 56-87 88-96  97-106 1-30 31-35 36-49 50-66 67-98 99-118 119-129259 204 205 1-22 23-33 34-48 49-55 56-87 88-96  97-106 1-30 31-35 36-4950-66 67-98 99-114 115-125 260 206 207 1-23 24-34 35-49 50-56 57-8889-98  99-108 1-30 31-35 36-49 50-66 67-98 99-114 115-125 261 208 2091-23 24-35 36-50 51-57 58-89 90-98  99-108 1-30 31-35 36-49 50-66 67-9899-113 114-124 262 210 211 1-23 24-34 35-49 50-56 57-88 89-98  99-1081-30 31-35 36-49 50-66 67-98 99-108 109-119 263 212 213 1-23 24-34 35-4950-56 57-88 89-98  99-108 1-30 31-35 36-49 50-66 67-98 99-112 113-123264 214 215 1-23 24-34 35-49 50-56 57-88 89-98  99-108 1-30 31-35 36-4950-66 67-98 99-109 110-120 265 216 217 1-23 24-34 35-49 50-56 57-8889-97  98-107 1-30 31-35 36-49 50-66 67-98 99-113 114-124 266 218 2191-23 24-34 35-49 50-56 57-88 89-99 100-109 1-30 31-35 36-49 50-66 67-9899-108 109-119 267 220 221 1-23 24-34 35-49 50-56 57-88 89-97  98-1071-30 31-35 36-49 50-66 67-98 99-106 107-117 270 222 223 1-23 24-39 40-5455-61 62-93  94-102 103-112 1-30 31-35 36-49 50-66 67-98 99-112 113-123271 224 225 1-23 24-39 40-54 55-61 62-93  94-102 103-112 1-30 31-3536-49 50-66 67-98 99-112 113-123 272 226 227 1-23 24-39 40-54 55-6162-93  94-102 103-112 1-30 31-35 36-49 50-66 67-98 99-113 114-124 273228 229 1-23 24-34 35-49 50-56 57-88 89-97  98-107 1-30 31-35 36-4950-66 67-98 99-105 106-116 274 230 231 1-23 24-39 40-54 55-61 62-93 94-102 103-112 1-30 31-35 36-49 50-66 67-98 99-112 113-123 275 232 2331-23 24-39 40-54 55-61 62-93  94-102 103-112 1-30 31-35 36-49 50-6667-98 99-105 106-138 276 234 235 1-23 24-34 35-49 50-56 57-88 89-97 98-107 1-30 31-35 36-49 50-66 67-98 99-105 106-116 277 236 237 1-2324-34 35-49 50-56 57-88 89-97  98-107 1-30 31-35 36-49 50-66 67-9899-105 106-116 278 238 239 1-23 24-39 40-54 55-61 62-93  94-102 103-1121-30 31-35 36-49 50-66 67-98 99-112 113-123 279 240 241 1-22 23-36 37-5152-58 59-90  91-102 103-112 1-30 31-35 36-49 50-66 67-98 99-112 113-123280 242 243 1-23 24-39 40-54 55-61 62-93  94-102 103-112 1-30 31-3536-49 50-66 67-98 99-112 113-123 281 244 245 1-23 24-39 40-54 55-6162-93  94-102 103-112 1-30 31-35 36-49 50-66 67-98 99-112 113-123 282246 247 1-23 24-39 40-54 55-61 62-93  94-102 103-112 1-30 31-35 36-4950-66 67-98 99-112 113-123 283 248 249 1-22 23-36 37-51 52-58 59-90 91-102 103-112 1-30 31-35 36-49 50-66 67-98 99-112 113-123 284 250 2511-22 23-36 37-51 52-58 59-90  91-102 103-112 1-30 31-35 36-49 50-6667-98 99-112 113-123 285 252 253 1-23 24-39 40-54 55-61 62-93  94-102103-112 1-30 31-35 36-49 50-66 67-98 99-112 113-123 287 254 255 1-2324-39 40-54 55-61 62-93  94-102 103-112 1-30 31-35 36-49 50-66 67-9899-113 114-124 288 256 257 1-22 23-36 37-51 52-58 59-90  91-102 103-1121-30 31-35 36-49 50-66 67-98 99-112 113-123 289 258 259 1-23 24-39 40-5455-61 62-93  94-102 103-112 1-30 31-35 36-49 50-66 67-98 99-112 113-123290 260 none 1-23 24-34 35-49 50-56 57-88 89-97  98-107 — — — — — — —298 none 261 — — — — — — — 1-30 31-35 36-49 50-66 67-98 99-115 116-126299 262 none 1-23 24-34 35-49 50-56 57-88 89-97  98-107 — — — — — — —301 263 none 1-23 24-34 35-49 50-56 57-88 89-97  98-107 — — — — — — —304 264 265 1-22 23-36 37-51 52-58 59-90  91-102 103-112 1-30 31-3536-49 50-66 67-98 99-112 113-123 305 266 267 or 1-22 23-36 37-51 52-5859-90  91-102 103-112 1-30 31-35 36-49 50-66 67-98 99-112 113-123 268

Example 3 Bispecific Antibodies that Bind IL-17A or IL-17F and the p19Subunit of IL-23

Bispecific antibodies can be constructed from the variable heavy andvariable light regions of antibodies that cross-bind to IL-17A andIL-17F with variable heavy and variable light regions of antibodies thatbind the p19 subunit of IL-23.

As an example, the antibodies that bind to IL-17A or to IL-17F, whichare produced by the ATCC deposited hybridomas described in Example 1 canbe joined with the variable heavy and variable light regions of theanti-IL-23p19 antibodies described in Example 2. Such bispecificantibodies can be joined in a variety of different configurations andcan comprise tandem scFv molecules (herein referred to as “tascFv”), andscFv molecules that are not tandem (herein referred to as “biscFv” and“BiAb”).

For the tascFv molecule, two scFv molecules are constructed such thatone scFv is amino terminal to the other one in a tandem configuration.This can be done in each orientation, and use a tether (e.g., a lambdastump tether or a CH1 stump tether, both of which are derived from thenative sequence just after the V region in the Fab, or a Gly-Sertether). The tascFv can be further constructed as fusion protein tocontain a Fc component (“tascFv Fc”). Thus the anti-IL-17A oranti-IL-17F binding entity can be the scFv entity that is eitherproximal or distal to a Fc component. Likewise, an anti-IL-23 bindingentity can be the scFv entity that is either proximal or distal to a Fccomponent.

The biscFv molecule is not a tandem configuration. Rather, it has a scFvat the N terminus and another at the C terminus of an Fc (“biscFv Fc”).These molecules can be made with the N terminal scFv directly fused tothe Fc hinge and with either a short or a long linker at the C terminusconnecting to the second scFv. These linkers are Gly-Ser. Thus, ananti-IL-17A or anti-IL-17F binding entity can be the scFv entity that iseither at the N terminus or at the C terminus to a Fc component.Likewise, an anti-IL-23 binding entity can be the scFv entity that iseither at the N terminus or at the C terminus to a Fc component.

The Biab molecule is also not a tandem format. It comprises of amonoclonal antibody with a scFv fused to the C terminus of the heavychain. These molecules can be made by converting one scFv back to alight chain (kappa or lambda) and a gamma1 heavy chain with the secondscFv connected by either a short or long Gly-Ser linker. Thus ananti-IL-17A or anti-IL-17F binding entity can be either scFv that isconverted back to a light chain (kappa or lambda) and a gamma1 heavychain or the second scFv fused to the C terminus. Likewise, ananti-IL-23 binding entity can be either scFv that is converted back to alight chain (kappa or lambda) and a gamma1 heavy chain or the secondscFv fused to the C terminus. Also, a Fab (either anti-IL-17A Fab,anti-IL-17F Fab or anti-IL-23 binding Fab) can be fused to the Fcportion rather than converting a scFv back to a light chain (kappa orlambda) and a gamma1 heavy chain.

Additional bispecific molecules are known in the art and include singlevariable domain antibodies, camelid antibodies, variable domains fusedto human serum albumin (See Muller, D, et al., J. Biol. Chem. 282 (Issue17):12650-12660, 2007), and dual variable domain immunoglobin molecules(See WIPO patent publication number WO/2007/024715, published Mar. 1,2007, by Wu, Chengbin, et al.), “Knob-into-hole” configurationsdescribed by Carter, et al. (1996); “IgG-C-terminal scFv” configurationdescribed by Morrison, et al. (1997); “Tandem scFv-Fc” configurationdescribed by Kanner, et al. (1998); “Diabody-Fc” configuration describedby Kontermann, et al. (1999); “scFv-Fc-scFv” configuration described byBarbas, et al. (2003).

Example 4 IL-17A/F mAb Competitive Binding Assay Protocol

To assess the ability of the anti-IL-17A/anti-IL-17F cross-bindingantibodies of the present invention to bind the ligands IL-17A andIL-17F, a Flow Cytometry-based competitive binding assay is utilized.Incubation of a BHK cell line stably transfected with full lengthIL-17RC in the presence of the ligands IL-17A or IL-17F, and an IL-17A/Fantibody of the present invention targeted to bind the ligands allowsfor detection and relative quantification of ligand bound to the cellsurface (and therefore unbound by the antibody). The biotinylation ofthe ligand allows for FACS detection using a secondary Streptavidinconjugated fluorophore. A reduction in cell bound ligand over atitration of the antibody is recorded as a reduction in the meanfluorescence of the cells.

Biotinylated ligands are individually pre-mixed at 1 ug/ml withtitrating amounts of antibody in staining media (HBSS+1% BSA+0.1%NaAzide+10 mM HEPES) in 100 ul volumes and incubated at RT for 15minutes. A BHK cell line stably transfected with full length IL17RC isprepared for ligand staining by resuspension with Versene (Invitrogencat. 15040-066), equilibrating to 2×10e5 cells/100 ul, pelleting, andresuspension in the ligand/antibody pre-mix. Stained cells are incubatedat 4° for 30 minutes, washed 1× in staining media, and stained withStreptavidin-PE (BD Pharmingen cat. 554061) at a 1:100 ratio. Cells areincubated at 4° in the dark for 30 minutes, washed 2× in staining media,and re-suspended in a 1:1 ratio of staining media and Cytofix (BDBioscience 554655). The BD LSRII Flow Cytometer or similar instrument isused for data collection and analysis. The software calculates the IC50for each curve. Antibodies having an IC50 value similar to those of theATCC Patent Deposit Designation PTA-7987 (clone 339.15.5.3), ATCC PatentDeposit Designation PTA-7988 (clone 339.15.3.6), and ATCC Patent DepositDesignation PTA-7989 (clone 339.15.6.16), will be effective atinhibiting, reducing, or neutralizing the effects of IL-17A or IL-17Fand thus be useful in methods for inhibiting inflammation in a mammalcomprising administering an antagonist of IL-23 and an antagonist ofIL-17A or IL-17F to the mammal, wherein the antagonist of IL-17A orIL-17F can bind IL-17A or IL-17F. In this assay, the IC50 (ug/ml)against IL-17A was between 28 and 38 (i.e., 28 ug/ml, 35 ug/ml, and 38ug/ml). In this assay the IC50 against IL-17F was between 3.5 and 3.6ug/ml. IC50 values against IL-17A and IL-17F between 2 ug/ml and 380ug/ml are contemplated by this invention.

Example 5 Inhibition of Activation by Human IL-17A and Human IL-17AF inMurine Nih3t3 Cells Using an Antagonist to Human IL-17A or IL-17F

A murine nih3t3 cell line was stably transfected with the kz170 (nfkb)reporter construct, containing a neomycin-selectable marker. See U.S.patent application Ser. No. 11/762,738, filed Jun. 13, 2007. This cellline, or a similar one, can be used to determine the EC50 levels of theantibodies that bind to IL-17A or IL-17F.

Antibodies to human IL-17A are used as antagonists of human IL-17A orhuman IL-17AF activation of nfkb elements in a luciferase assay. In thisassay, EC50 levels of human IL-17A- or IL-17AF-mediated nfkb activationin the murine nih3t3/kz170 assay cell line is measured. For highlyeffective antibodies, when used at approx. 10 μg/mL concentration, theantibody can completely neutralize activity induced by human IL-17A orIL-17AF, with the inhibition of activity decreasing in a dose dependentfashion at the lower concentrations. An isotype-matched negative controlmAb, tests at the concentrations described above, provided no inhibitionof activity. These results demonstrate that antibodies against IL-17A orIL-17F are able to antagonize the activity of the pro-inflammatorycytokines, IL-17A and IL-17AF.

Example 6 Bioassay for Neutralization of huIL-17-Induced CytokineProduction in Human Small Airway Epithelial Cells (SAEC IL-12 PHABioassay)

Treatment of human small airway epithelial cells (SAEC) with rhIL-17induces the production of cytokines G-CSF, IL-6, and IL-8, which inturn, play a role in the pathology associated with the diseases forwhich a bispecific neutralizing antibody comprising an antibody orantibody fragment that cross-binds IL-17A and IL-17F and an antibody orantibody fragment that binds IL-23p19 would be efficacious. The abilityof any of the neutralizing entities described herein to inhibitIL-17-mediated production of these cytokines is measured in thisbioassay, thus being predictive of in vivo efficacy against thesecytokines as well.

Method: SAEC (cells and growth media purchased from Cambrex, Inc.) areplated at 8,000 cells/well in 96-well flat bottom tissue culturemulti-well plates, and placed in a 370 C, 5% CO2 incubator. Thefollowing day, cells are treated with a dose range of the neutralizingentity in combination with 10-20 ng/mL rhIL-17. The ligand andneutralizing entity are incubated together for 30 minutes at 370 Cbefore adding to the cells. Duplicate or triplicate wells are set up foreach dose. After 24-48 hours, supernatants are collected, and stored at−800 C if not used directly. Before taking supernatants, wells arescanned by inverted microscope to make note of which wells hadconsiderable cell death. Those wells are not included in the finalcalculations. Supernatants are then assayed for cytokines huG-CSF,huIL-6, and huIL-8 in a multiplex bead-based assay system (Bio-RadLaboratories), and IC50 determined.

In the presence of rhIL-17, antibodies that cross-bind to IL-17A orIL-17F are efficacious at reducing cytokine production with IC50 valuesranging from 0.1-100 nM.

Example 7 Bioassay for Neutralization of huIL17A-Induced G-CSF and IL-6Cytokine Production in U373MG and U87MG Human Glioblastoma Cells

rhIL-17A treatment induces the production of cytokine IL-6 in humanglioblastoma cells U373MG, and of cytokines G-CSF and IL-6 in humanglioblastoma cells U87MG. The cell lines are available from commercialvendors such as ATCC (Manassas, Va.). These cytokines, in turn, play arole in the pathology associated with the diseases for which antagonistsof IL-17A and IL23p19 would be efficacious. The ability of any of theantagonists described herein to inhibit rhIL-17A-mediated production ofG-CSF and IL-6 is measured in this bioassay, thus being predictive of invivo efficacy against this cytokine as well.

Method: Cells are plated in media (MEM w/Earle's salts, 10% FCS, 2 mML-glutamine, 1 mM sodium pyruvate, 100 uM NEAA) at 3,000-7,000cells/well in 96-well flat bottom tissue culture multi-well plates andplaced in a 37° C., 5% CO2 incubator from 1 hr to overnight. Doses ofthe neutralizing entities are prepared in culture media with FCSconcentration reduced to 2%. The cells are treated with a dose range ofthe neutralizing entity in combination with 0.20-0.5 nM rhIL-17A. Theligand and neutralizing entity are incubated together for 30 minutes at37° C. before adding to the cells. Supernatants are collected after 24hours and assayed for huG-CSF and huIL-6 using a bead-based assay system(Bio-Rad Laboratories), and IC50 determined.

In the presence of rhIL-17A, the anti-IL-17A antagonists are efficaciousat reducing huIL-6 cytokine production by U373MG cells, with IC50 valuesranging from 2.3-45 nM. For U87MG cells, neutralization of huG-CSFcytokine production with IC50 values ranging from 0.17-0.52 nM isefficacious.

Example 8 IL-12 Bioassy

Leukopheresis PBMC: To obtain a consistent pool of PBMC's, normal humandonors are voluntarily apheresed. The leukopheresis PBMC are poured intoa sterile 500 ml plastic bottle, diluted to 400 ml with room temperaturePBS+1 mM EDTA and transferred to 250 ml conical tubes. The 250 ml tubesare centrifuged at 1500 rpm for 10 minutes to pellet the cells. The cellsupernatant is then removed and discarded. The cell pellets are thencombined and suspended in 400 ml PBS+1 mM EDTA. The cell suspension (25ml/tube) is overlaid onto Ficoll (20 ml/tube) in 50 ml conical tubes.The tubes are centrifuged at 2000 rpm for 20 minutes at roomtemperature. The interface layer (“buffy coat”) containing the whiteblood cells and residual platelets is collected, pooled and washedrepeatedly with PBS+1 mM EDTA until the majority of the platelets areremoved. The white blood cells are suspended in 100 ml of ice-coldCryopreservation medium (70% RPMI+20% FCS+10% DMSO) and distributed intosterile cryovials (1 ml cells/vial). The cryovials are placed in a −80°C. freezer for 24 hours before transfer to a liquid-nitrogen freezer.The white blood-cell yield from a typical apheresis is 0.5-1.0×1010cells. Apheresis cells processed in this manner contain T cells, Bcells, NK cells, monocytes and dendritic cells.

Preparation of PHA blasts: T cells must be activated in order to expressthe IL-12 receptor and be able to respond to IL-12 and IL-23.Cryopreserved leukopheresis PBMC are thawed, transferred to a sterile 50ml conical tube, washed once with 50 ml of warm RPMI+10%heat-inactivated FBS+1 ug/ml DNAse I (Calbiochem), resuspended in 50 mlof fresh RPMI/FBS/DNAse medium and incubated in a 37° C. water bath forat least 1 hour to allow the cells to recover from being thawed. Thecells are then centrifuged and the cell-supernatant discarded. The cellpellet is resuspended in RPMI+10% FBS and distributed into sterile 75cm2 tissue culture flasks (1×107 cells/flask in 40 ml/flask). PHA-L (5mg/ml stock in PBS) is added to the cells at a final concentration of 5ug/ml. The cells are then cultured at 37° C. in a humidified incubatorfor a total of 5 days. The cells are “rested” for some experiments byharvesting the cells on the afternoon of day 4, replacing the culturemedium with fresh RPMI+10% FBS without PHA-L (40 ml/flask) and returningthe cells to their flasks and incubating at 37° C. the cells in ahumidified incubator for the remainder of the 5 day culture period.

IL-12 and IL1-23 bioassays: Three in vitro assays for detection of humanIL-12 and Il-23 bioactivity on normal human T cells have beenestablished: 1) IFN-gamma and MIP-1alpha production, 2) proliferation([3H]-incorporation) and 3) STAT3 activation. Human PHA blasts(activated T cells) are harvested on day 5 of culture, suspended infresh RPMI+10% FBS and plated at the desired cell number per well in 96well plates.

The inclusion of an IL-12 assay is used to determine specificity of theneutralizing entities described herein for IL-23p19 and not IL-12.

For the IFN-gamma production assay, the cells are plated at 1×106/wellin flat-bottom 96-well plates. The cells are cultured at 37° C. in afinal volume of 200 ul/well with either medium alone, human IL-2 alone(10 ng/ml; R & D Systems), human IL-12 alone (graded doses; Invitrogen),human IL-23 alone (graded doses; made in-house; CHO-derived), anti-humanCD28 mAb alone (graded doses; clone 28.2, e-Biosciences), or eachcytokine in combination with anti-human CD28 mAb. Triplicate wells areset up for each culture condition. For the IFN-gamma production assay,cell supernatants (120 ul/well) are harvested after 24-48 hours ofculturing the cells at 37° C. in a humidified incubator. Human IFN-gammaand MIP-1alpha concentrations in these supernatants (pooled for eachtriplicate) are measured using a commercial Luminex bead-based ELISA kit(Invitrogen) following the manufacturer's instructions.

Effects of IL-23 on IFN-gamma and MIP-1alpha production are enhanced byculturing the cells with plate-immobilized anti-human CD3 mAb (5 ug/ml)and soluble anti-human CD28mAb (1 ug/ml) as well as harvesting thesupernatants (120 ul/well) after 48 hrs of culture at 37° C. the cellsin a humidified incubator. Human IFN-gamma concentrations in thesesupernatants (pooled for each triplicate) are measured using acommercial Luminex bead-based ELISA kit (Invitrogen) following themanufacturer's instructions.

For the [3H]-incorporation assay the cells are plated at 2×105cells/well in U-bottom 96-well plates. The cells are cultured at 37degrees C. for 72 hours. The cells are pulsed with 1 uCi/well of[3H]-Thymidine (Amersham) for the last 8 hours of this culture period.The cells are then harvested onto glass-fiber filters and the CPMs of[3H] incorporated are quantitated using a beta counter (Topcount NXT,Packard).

For each of these above endpoint parameters, effective neutralization ofactivity mediated by IL-23 is observed in the presence of anti-IL23p19neutralizing entities described herein at IC50 values that range from0.1 to ˜100 nM. No effect of the anti-IL-23p19 antagonists onneutralizing the effects mediated by IL-12, indicates specificity of theantagonists to IL-23p19.

STAT3 Bioassay: For the STAT3 Bioassay the cells are plated at 2×105cells/well in U-bottom 96-well plates. Serial dilutions of human IL-12(R&D) or recombinant human IL-23 (in-house CHO-derived material oreBioscience's Insect heterodimer material) are prepared in assay media(RPMI 1640 with L-Glutamine plus 10% fetal bovine serum), added to theplates containing the cells and incubated together at 37° C. for 15minutes. Additionally, the assay is also used to measure neutralizationof IL-12 and IL-23 activity using either commercially-availableneutralizing reagents (as “controls”) or the anti-IL-23p19-containingneutralizing entities described herein. A half-maximal concentration(EC50, effective concentration at 50 percent) of IL-12 or IL-23 arecombined with serial dilutions of anti-human IL-12 p40 monoclonalantibody (Pharmingen), anti-human IL-23 p19 polyclonal antibody (R&D,AF1716), human IL-23R-Fc Soluble Receptor, or any of the neutralizingentities described herein, and incubated together at 37° C. for 30minutes in assay media prior to addition to cells. Followingpre-incubation, treatments are added to the plates containing the cellsand incubated together at 37° C. for 15 minutes.

Following incubation, cells are washed with ice-cold wash buffer and puton ice to stop the reaction, according to manufacturer's instructions(BIO-PLEX Cell Lysis Kit, BIO-RAD Laboratories, Hercules, Calif.). Cellsare then spun down at 2000 rpm at 4° C. for 5 minutes prior to removingthe media. 50 ul/well lysis buffer is added to each well; lysates arepipetted up and down five times while on ice, then agitated on amicroplate platform shaker for 20 minutes at 300 rpm and 4° C. Platesare centrifuged at 4500 rpm at 4° C. for 20 minutes. Supernatants arecollected and transferred to a new micro titer plate for storage at −20°C.

Capture beads (BIO-PLEX Phospho-STAT3 Assay, BIO-RAD Laboratories) arecombined with 50 ul of 1:1 diluted lysates and added to a 96-well filterplate according to manufacture's instructions (BIO-PLEX PhosphoproteinDetection Kit, BIO-RAD Laboratories). The aluminum foil-covered plate isincubated overnight at room temperature, with shaking at 300 rpm. Theplate is transferred to a microtiter vacuum apparatus and washed threetimes with wash buffer. After addition of 25 μL/well detection antibody,the foil-covered plate is incubated at room temperature for 30 minuteswith shaking at 300 rpm. The plate is filtered and washed three timeswith wash buffer. Streptavidin-PE (50 ul/well) was added, and thefoil-covered plate is incubated at room temperature for 15 minutes withshaking at 300 rpm. The plate is filtered and washed two times with beadresuspension buffer. After the final wash, beads are resuspended in 125ul/well of bead suspension buffer, shaken for 30 seconds, and read on anarray reader (BIO-PLEX, BIO-RAD Laboratories) according to themanufacture's instructions. Data are analyzed using analytical software(BIO-PLEX MANAGER 3.0, BIO-RAD Laboratories).

Increases in the level of the phosphorylated STAT3 transcription factorpresent in the lysates are indicative of an IL-12 or IL-23receptor-ligand interaction. For the neutralization assay, decreases inthe level of the phosphorylated STAT3 transcription factor present inthe lysates are indicative of neutralization of the IL-12 or IL-23receptor-ligand interaction. IC50 (inhibitory concentration at 50percent) values are calculated using GraphPad Prism®4 software (GraphPadSoftware, Inc., San Diego Calif.) and expressed as molar ratios for eachreagent and/or neutralizing entity in the neutralization assay.

Efficacious anti-IL-23p19 neutralizing entities are equally or betterthan the commercially available reagents at neutralizing the effects ofrhIL-23 and they specifically inhibit rhIL-23 and not IL-12.

Example 9 IL-12 Bioassy Bioassay for Neutralization of Human IL-23Mediated IL-17A and IL-17F Production in Murine Splenocytes

Recombinant human IL-23 (rhIL-23) induces the production of IL-17A andIL-17F in murine splenocytes. To evaluate antagonists to IL-23,neutralization of IL-17A and IL-17F production in rhIL-23 treated murinesplenocytes is examined. Antagonists to rhIL-23 are compared to thecommercial neutralizing antibody anti-IL-12p40 (Pharmingen, FranklinLakes, N.J.).

Experimental protocol: A single cell suspension of splenocytes areprepared from whole spleens harvested from either C57BL/6 or BALB/cmice. After red blood cell lysis with ACK buffer (0.010 M KHCO3, 0.0001M EDTA, 0.150 M NH4Cl), splenocytes are washed and resuspended in RPMIbuffer (containing 1% non-essential amino acids, 1% Sodium Pyruvate, 2.5mM HEPES, 1% L-glutamine, 0.00035% 2-mercaptoethanol, 1% Pen/Strep, 10%FCS and 50 ng/ml human IL-2 (R&D Systems, Minneapolis, Minn.)). Cellsare seeded at 500,000 cells per well in a 96-well round bottom plate. Ina separate plate, rhIL-23 at a concentration of 10 pM is pre-incubatedfor 30-90 minutes at 37° C. with 3-fold serial dilutions of theantagonists. Concentrations of the antagonists range from 0-343 nM. TheIL-23 ligand plus antagonists are then added to the splenocytes andincubated at 37° C., 5% CO2 for 24-72 hours. The supernatants arecollected and frozen at −80° C. until ready to process. The levels ofIL-17A and IL-17F protein in the supernatants are measured usingbead-based sandwich ELISAs. A commercial kit (Upstate, Charlottesville,Va.) is used to measure IL-17A protein. A bead-based ELISA developedin-house using an antibody to IL-17F (R&D) conjugated to a bead is usedto measure IL-17F. IC50 values for each antagonist are calculated as theamount of antagonist needed to neutralize 50% of the activity ofrhIL-23.

In the presence of rhIL-23, the anti-IL-23p19 antibodies are efficaciousat reducing IL-17A and IL-17F production with IC50 values in the rangeof 0.27-100.0 nM.

Example 10 Disease Incidence and Progression in Mouse ExperimentalAllergic Encephalomyelitis (EAE) as a Model of Multiple SclerosisRecombinant Human IL-23 (rhIL-23

A) Mouse Allergic Encephalomyelitis (EAE) Model

To study mechanism and evaluate the effects of potential therapies formultiple sclerosis, the animal model of experimental autoimmuneencephalomyelitis (EAE) is commonly used. For the relapsing-remittingEAE model, 9 to 10 week old female SJL mice (Jackson or Charles RiverLabs) are immunized subcutaneously with proteolipid peptide (PLP)emulsified in complete Freund's adjuvant, and with intravenous pertussistoxin. Within approximately 6 to 23 days, animals begin to show symptomsof weight loss and paralysis that are characteristic of this model. Theextent of disease is evaluated daily in the mice by taking their bodyweights and assigning a clinical score (0-8) to each mouse, as detailedbelow. The typical pattern of disease symptoms in immunized, butotherwise untreated mice, is one of weight loss and paralysis, followedby a period of disease symptom remission, and a subsequent relapse ofdisease symptoms. A pattern of relapses and remissions of diseasesymptoms ensues, which is also found in humans with this type ofmultiple sclerosis, known as relapsing-remitting disease. Chronicprogressive and secondary progressive multiple sclerosis are alsotargeted indications for this therapeutic combination of an antibodythat binds IL-17A or IL-17F and IL-23/p19 such as a bispecific antibodyor scFV as described in this invention. These latter types of multiplesclerosis are tested in a similar manner using MOG35-55 peptide inC57BL/6 mice, instead of PLP in SJL mice.

Neutralizing monoclonal antibodies to mouse IL-17A and IL-23p19 areadministered separately or as a therapeutic combination, duringremission from the first peak of EAE disease symptoms. The antibodiesare delivered as intraperitoneal injections every other day, or as asimilar dosing regimen. Groups receive either 25, 50 or 100 ug of eachantibody, alone or as a therapeutic combination, per animal per dose,and control groups receive the vehicle control, PBS (Life Technologies,Rockville, Md.) or antibody isotype control.

B) Monitoring Disease

Animals can begin to show signs of paralysis and weight loss betweenapproximately 6 and 23 days following PLP or MOG35-55 immunizations.Most animals develop symptoms within 11-17 days of the immunizations,but some may show symptoms sooner or later than this.

All animals are observed, weighed, and assigned a clinical score dailyto assess the status of disease.

C) Clinical Score

Clinical Score is measured as follows: 0=Normal; healthy; 1=slight tailweakness (tip of tail does not curl and); 2=tail paralysis (unable tohold tail upright); 3=tail paralysis and mild waddle; 4=tail paralysisand severe waddle; 5=tail paralysis and paralysis of one limb; 6=tailparalysis and paralysis of any 2 limbs; 7=tetraparesis (all 4 limbsparalysed); and 8=moribund or dead

Blood is collected throughout the experiment to monitor serum levels ofcytokine and levels of other mediators of disease. At the time ofeuthanasia, blood is collected for serum, and brain and spinal cordcollected in 10% NBF for histology. In separate animals, tissues(including lymph nodes, brain, spinal cord, spleen, and others) areharvested for the quantification of mRNA by TaqMan quantitativereal-time PCR.

D) Results

Groups of mice (n=13-15 each) receiving the therapeutic combination ofneutralizing monoclonal antibodies to IL-17 and IL-23/p19 arecharacterized by a significant (p<0.05) reduction in disease severity asevidenced by significant (p<0.05) reductions in clinical score and bodyweight loss compared to mice treated with PBS, either of the antibodiesalone at similar doses as those used in the combination, or isotypecontrol antibodies. Furthermore, the mice treated with the therapeuticantibody combination, i.e., an antibody that binds IL-17A or IL-17F andIL-23p19 may show a complete absence of disease relapse.

Significant reductions in serum IL-6, IL-13, IL-17A, IL-23, G-CSF, andTNF-α concentrations compared to PBS-treated mice will also beefficacious. Samples are collected at the same time point following peakof first disease onset and after the same number of antibody doses.Draining lymph nodes are harvested from the mice at this same time pointand cultured for 24 h with PLP139-151.

Thus the therapeutic combination of an antibody that binds IL-17A orIL-17F and IL23/p19 may be more efficacious in the treatment of EAE as amodel of human multiple sclerosis. The therapeutic combination canreduce clinical disease symptoms and works at the molecular level toreduce inflammation, inflammatory infiltrates, inflammatorycytokines/chemokines, and other mechanisms known to be affected in thismanner.

Example 11 Disease Incidence and Progression in Mouse Murine Colitis

IL-23 and IL-17 are important players in murine colitis and human IBD,via the actions of Th17 cells. IL-23 and IL-17 are upregulated incolitis and IBD, and neutralization of either cytokine alone isefficacious in several animal models of colitis (Fujino et al, Gut,2003, 52:65-70; Schmidt et al, Inflamm Bowel Dis. 2005, 11:16-23; Yen etal, J Clin Invest. 2006, 116:1310-1316; Zhang et al, Inflamm Bowel Dis.2006, 12:382-388; Kullberg et al, J Exp Med. 2006, 203:2485-94.). SinceIL-23 is important for the maintenance, differentiation, and/orinduction of Th17 cells, neutralization of both cytokines would be moreefficacious at reducing disease than either cytokine alone.

Methods: For this experiment, 40 C57BL/10 female mice (obtained fromHarlan) are used. On day −5, mice are treated topically with 200 ul of3.0% (w/v) oxazalone in 100% ethanol (“sensitization”) on the abdomen.On day 0, all mice receive intrarectal injections (120 uL each) of 2.0%(w/v) oxazalone in 50% ethanol while under light isoflurane gasanesthesia (“challenge”). Mice are monitored for disease using a DiseaseActivity Index (DAI) score, which includes stool consistency, bodyweight, and blood in stool. For mAb treatments, mice are administeredone of the following, via i.p. injection on days −5, −3, and −1: PBS, 50ug neutralizing anti-mouse IL-17, 50 ug neutralizing anti-mouse IL-23p19mAb, or a combination of the anti-IL17+IL-23p19 mAb's.

Mice are euthanized on day 2. Serum is collected and stored for lateranalysis; colons are removed and observed for any gross signs of colitis(lesion, colon shortening, and colon wall thickening). Colons are thencut longitudinally and processed for histology and for 24 h coloncultures.

A significant reduction in DAI score and significant improvement inhistological morphology (e.g. reduced colonic damage and reducedinflammation, shortened colon) in mice treated with the combination ofanti-IL-17+anti-IL-23p19 antibodies, compared to PBS and either mAbalone would show efficacious treatment.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

What is claimed is:
 1. A method of treating an inflammatory disease in ahuman patient comprising subcutaneously or intravenously administeringto the patient a therapeutically effective amount of a bispecificantibody comprising a cross-reactive IL-17A and IL-17F binding entityand an IL-23p19 binding entity, wherein the cross-reactive IL-17A andIL-17F binding entity comprises a humanized antibody derived from theantibody produced by the hybridoma deposited with the American TypeCulture Collection having the ATCC Patent Deposit Designation PTA-7988,wherein the IL-23p19 binding entity comprises an antibody whichspecifically binds to the p19 subunit of IL-23, and wherein theinflammatory disease is psoriasis or psoriatic arthritis.
 2. The methodof claim 1, wherein the isotype of the bispecific antibody is IgG.
 3. Amethod of treating an inflammatory disease in a human patient comprisingsubcutaneously or intravenously administering to the patient atherapeutically effective amount of a composition comprising abispecific antibody and a pharmaceutically acceptable carrier ordiluent, wherein the bispecific antibody comprises a cross-reactiveIL-17A and IL-17F binding entity and an IL-23p19 binding entity, whereinthe cross-reactive IL-17A and IL-17F binding entity comprises ahumanized antibody derived from the antibody produced by the hybridomadeposited with the American Type Culture Collection having the ATCCPatent Deposit Designation PTA-7988, wherein the IL-23p19 binding entitycomprises an antibody which specifically binds to the p19 subunit ofIL-23, and wherein the inflammatory disease is psoriasis or psoriaticarthritis.
 4. The method of claim 3, wherein the isotype of thebispecific antibody is IgG.